RESUMO
The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Simulação por Computador , Desenho de Fármacos , SARS-CoV-2 , Animais , Feminino , Humanos , Camundongos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Mutação , Testes de Neutralização , SARS-CoV-2/classificação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Análise Mutacional de DNA , Deriva e Deslocamento Antigênicos/genética , Deriva e Deslocamento Antigênicos/imunologia , Desenho de Fármacos/métodosRESUMO
SUMMARY: The design of two overlapping genes in a microbial genome is an emerging technique for adding more reliable control mechanisms in engineered organisms for increased stability. The design of functional overlapping gene pairs is a challenging procedure, and computational design tools are used to improve the efficiency to deploy successful designs in genetically engineered systems. GENTANGLE (Gene Tuples ArraNGed in overLapping Elements) is a high-performance containerized pipeline for the computational design of two overlapping genes translated in different reading frames of the genome. This new software package can be used to design and test gene entanglements for microbial engineering projects using arbitrary sets of user-specified gene pairs. AVAILABILITY AND IMPLEMENTATION: The GENTANGLE source code and its submodules are freely available on GitHub at https://github.com/BiosecSFA/gentangle. The DATANGLE (DATA for genTANGLE) repository contains related data and results and is freely available on GitHub at https://github.com/BiosecSFA/datangle. The GENTANGLE container is freely available on Singularity Cloud Library at https://cloud.sylabs.io/library/khyox/gentangle/gentangle.sif. The GENTANGLE repository wiki (https://github.com/BiosecSFA/gentangle/wiki), website (https://biosecsfa.github.io/gentangle/), and user manual contain detailed instructions on how to use the different components of software and data, including examples and reproducing the results. The code is licensed under the GNU Affero General Public License version 3 (https://www.gnu.org/licenses/agpl.html).
Assuntos
Software , Biologia Computacional/métodos , Genoma Microbiano , Engenharia Genética/métodosRESUMO
The development of synthetic biological circuits that maintain functionality over application-relevant time scales remains a significant challenge. Here, we employed synthetic overlapping sequences in which one gene is encoded or 'entangled' entirely within an alternative reading frame of another gene. In this design, the toxin-encoding relE was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. A functional entanglement construct was obtained upon modification of the ribosome-binding site of the internal relE gene. Using this optimized design, we found that the selection pressure to maintain functional IlvA stabilized the production of burdensome RelE for >130 generations, which compares favorably with the most stable kill-switch circuits developed to date. This stabilizing effect was achieved through a complete alteration of the allowable landscape of mutations such that mutations inactivating the entangled genes were disfavored. Instead, the majority of lineages accumulated mutations within the regulatory region of ilvA. By reducing baseline relE expression, these more 'benign' mutations lowered circuit burden, which suppressed the accumulation of relE-inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement paired with an adaptive laboratory evolution campaign to increase the evolutionary stability of burdensome synthetic circuits.
Assuntos
Homologia de Genes , Engenharia Genética , Sítios de Ligação , Escherichia coli/genética , Mutação , Ribossomos/genética , Pseudomonas/genética , Engenharia Genética/métodosRESUMO
The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a ß-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.
Assuntos
Proteínas da Membrana Bacteriana Externa , Endopeptidases , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Membrana , Desdobramento de Proteína , Fatores de Transcrição , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Faithful cell cycle progression in the dimorphic bacterium Caulobacter crescentus requires spatiotemporal regulation of gene expression and cell pole differentiation. We discovered an essential DNA-associated protein, GapR, that is required for Caulobacter growth and asymmetric division. GapR interacts with adenine and thymine (AT)-rich chromosomal loci, associates with the promoter regions of cell cycle-regulated genes, and shares hundreds of recognition sites in common with known master regulators of cell cycle-dependent gene expression. GapR target loci are especially enriched in binding sites for the transcription factors GcrA and CtrA and overlap with nearly all of the binding sites for MucR1, a regulator that controls the establishment of swarmer cell fate. Despite constitutive synthesis, GapR accumulates preferentially in the swarmer compartment of the predivisional cell. Homologs of GapR, which are ubiquitous among the α-proteobacteria and are encoded on multiple bacteriophage genomes, also accumulate in the predivisional cell swarmer compartment when expressed in Caulobacter The Escherichia coli nucleoid-associated protein H-NS, like GapR, selectively associates with AT-rich DNA, yet it does not localize preferentially to the swarmer compartment when expressed exogenously in Caulobacter, suggesting that recognition of AT-rich DNA is not sufficient for the asymmetric accumulation of GapR. Further, GapR does not silence the expression of H-NS target genes when expressed in E. coli, suggesting that GapR and H-NS have distinct functions. We propose that Caulobacter has co-opted a nucleoid-associated protein with high AT recognition to serve as a mediator of cell cycle progression.
Assuntos
Sequência Rica em At/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Caulobacter crescentus/metabolismo , Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Alphaproteobacteria/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Caulobacter crescentus/genética , Ciclo Celular/genética , Divisão Celular/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Loci Gênicos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Frações Subcelulares/metabolismoRESUMO
Structural characterization of lipid A from Gram-negative bacteria remains a significant challenge, especially with respect to localizing modifications of the phosphate groups typically found on the reducing and non-reducing ends of the ß-1',6-linked glucosamine disaccharide backbone of lipid A. As reported here, combining traditional collisional activated dissociation (CAD) and ultraviolet photodissociation (UVPD) in a hybrid MS3 approach facilitates identification and localization of substituents of the phosphate groups. The focus is on rapid identification and characterization of substituted lipid A species with specific emphasis on the modifications on the 1 and 4' phosphate moieties. Mapping these modifications, typically ones that modify the surface charges of lipopolysaccharides, is particularly important owing to the impact of these types of modifications on antibiotic resistance. The presence of phosphoethanolamine, aminoarabinose, and galactosamine moieties in hexaacylated and heptaacylated lipid A species, including ones from Enterobacter cloacae and Acinetobacter baumannii, are characterized using a targeted MS3 strategy to identify glycosidic product ions (1,5X1 and 0,4A2, typically) which allow localization of the substituents.
RESUMO
Most integral outer membrane proteins (OMPs) of Gram-negative bacteria, such as Escherichia coli, assume a ß-barrel structure. The ß-barrel assembly machine (Bam), a five-member complex composed of ß-barrel OMP BamA and four associated lipoproteins, BamB, BamC, BamD, and BamE, folds and inserts OMPs into the outer membrane. The two essential proteins BamA and BamD interact to stabilize two subcomplexes, BamAB and BamCDE, and genetic and structural evidence suggests that interactions between BamA and BamD occur via an electrostatic interaction between a conserved aspartate residue in a periplasmic domain of BamA and a conserved arginine in BamD. In this work, we characterize charge-change mutations at these key BamA and BamD residues and nearby charged residues in BamA with respect to OMP assembly and Bam complex stability. We show that Bam complex stability does not correlate with function, that BamA and BamD must adopt at least two active conformational states during OMP assembly, and that these charged residues are not required for function. Rather, these charged residues are important for coordinating the activities of BamA and BamD to allow efficient OMP assembly. We present a model of OMP assembly wherein recognition and binding of unfolded OMP substrate by BamA and BamD induce a signaling interaction between the two proteins, causing conformational changes necessary for the assembly reaction to proceed. By analogy to signal sequence recognition by SecYEG, we believe these BamA-BamD interactions ensure that both substrate and complex are competent for OMP assembly before the assembly reaction commences.IMPORTANCE Conformational changes in the proteins of the ß-barrel assembly machine (Bam complex) are associated with the folding and assembly of outer membrane proteins (OMPs) in Gram-negative bacteria. We show that electrostatic interactions between the two essential proteins BamA and BamD coordinate conformational changes upon binding of unfolded substrate that allow the assembly reaction to proceed. Mutations affecting this interaction are lethal not because they destabilize the Bam complex but rather because they disrupt this coordination. Our model of BamA-BamD interactions regulating conformation in response to proper substrate interaction is reminiscent of conformational changes the secretory (Sec) machinery undergoes after signal sequence recognition that ensure protein quality control.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Multimerização Proteica , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Análise Mutacional de DNA , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Biológicos , Modelos Moleculares , Conformação ProteicaRESUMO
UNLABELLED: The biogenesis of the outer membrane (OM) of Escherichia coli is a conserved and vital process. The assembly of integral ß-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric ß-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or nonessential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, low-abundance, and difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple and abundant substrates that are not as dependent on SurA but are strongly dependent on BamB for assembly. Here, we describe novel mutations in bamD that lower levels of BamD 10-fold and >25-fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wild-type BamA levels, to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM. IMPORTANCE: The outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral ß-barrel proteins (OMPs) are assembled by the ß-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação , Dobramento de ProteínaRESUMO
UNLABELLED: The periplasmic chaperone SurA is critical for the biogenesis of outer membrane proteins (OMPs) and, thus, the maintenance of membrane integrity in Escherichia coli. The activity of this modular chaperone has been attributed to a core chaperone module, with only minor importance assigned to the two SurA peptidyl-prolyl isomerase (PPIase) domains. In this work, we used synthetic phenotypes and covalent tethering to demonstrate that the activity of SurA is regulated by its PPIase domains and, furthermore, that its activity is correlated with the conformational state of the chaperone. When combined with mutations in the ß-barrel assembly machine (BAM), SurA mutations resulting in deletion of the second parvulin domain (P2) inhibit OMP assembly, suggesting that P2 is involved in the regulation of SurA. The first parvulin domain (P1) potentiates this autoinhibition, as mutations that covalently tether the P1 domain to the core chaperone module severely impair OMP assembly. Furthermore, these inhibitory mutations negate the suppression of and biochemically stabilize the protein specified by a well-characterized gain-of-function mutation in P1, demonstrating that SurA cycles between distinct conformational and functional states during the OMP assembly process. IMPORTANCE: This work reveals the reversible autoinhibition of the SurA chaperone imposed by a heretofore underappreciated parvulin domain. Many ß-barrel-associated outer membrane (OM) virulence factors, including the P-pilus and type I fimbriae, rely on SurA for proper assembly; thus, a mechanistic understanding of SurA function and inhibition may facilitate antibiotic intervention against Gram-negative pathogens, such as uropathogenic Escherichia coli, E. coli O157:H7, Shigella, and Salmonella. In addition, SurA is important for the assembly of critical OM biogenesis factors, such as the lipopolysaccharide (LPS) transport machine, suggesting that specific targeting of SurA may provide a useful means to subvert the OM barrier.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/fisiologia , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/genética , Análise Mutacional de DNA , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Peptidilprolil Isomerase/genética , Conformação Proteica , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
In gram-negative bacteria, integral outer membrane ß-barrel proteins (OMPs) are assembled by the beta-barrel assembly machine (Bam) complex. The essential components of this complex are the OMP BamA [which contains a carboxyl-terminal ß-barrel and an amino-terminal periplasmic module composed of five polypeptide transport associated (POTRA) domains] and the lipoprotein BamD. In Escherichia coli, the Bam complex also contains three nonessential lipoproteins (BamBCE), all of which require the barrel-proximal POTRA domain (P5) for stable interactions with BamA. We have previously reported that the BamA ß-barrel assumes two different conformations. A method for conformation-specific labeling of BamA described here reveals that these conformers reflect the degree of surface exposure of the conserved sixth extracellular loop (L6). L6 is surface accessible in one conformation but not in the other, likely because it occupies the lumen of the BamA ß-barrel in the latter case. A gain-of-function mutation that promotes Bam activity (bamDR197L) and a loss-of-function mutation that decreases the activity of Bam (ΔbamE) both favor surface exposure of BamA L6, suggesting that BamD and BamE normally act to control L6 exposure through opposing functions. These results, along with the synthetic lethality of the bamDR197L ΔbamE double mutant, imply a cyclic mechanism in which the Bam lipoproteins regulate the conformation of BamA during the OMP assembly reaction. Our results further suggest that BamDE controls L6 exposure via conformational signals transmitted through P5 to L6.
Assuntos
Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
UNLABELLED: The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFa and rsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFa and RsaFb, which are homologues of TolC in Escherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaFa and rsaFb by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaFa mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFa and RsaFb in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus. IMPORTANCE: Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus provide significant insight into the mechanisms of U resistance by C. crescentus. In particular, we show that outer membrane transporters RsaFa and RsaFb, previously known as part of the S-layer export machinery, may confer U resistance by U efflux and/or by maintaining membrane integrity during U stress.
Assuntos
Caulobacter crescentus/metabolismo , Elementos de DNA Transponíveis/genética , Estresse Fisiológico/efeitos dos fármacos , Urânio/toxicidade , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Caulobacter crescentus/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Mutagênese , TranscriptomaRESUMO
The outer membrane (OM) of gram-negative bacteria such as Escherichia coli contains lipoproteins and integral ß-barrel proteins (outer-membrane proteins, OMPs) assembled into an asymmetrical lipid bilayer. Insertion of ß-barrel proteins into the OM is mediated by a protein complex that contains the OMP BamA and four associated lipoproteins (BamBCDE). The mechanism by which the Bam complex catalyzes the assembly of OMPs is not known. We report here the isolation and characterization of a temperature-sensitive lethal mutation, bamAE373K, which alters the fifth polypeptide transport-associated domain and disrupts the interaction between the BamAB and BamCDE subcomplexes. Suppressor mutations that map to codon 197 in bamD restore Bam complex function to wild-type levels. However, these suppressors do not restore the interaction between BamA and BamD; rather, they bypass the requirement for stable holocomplex formation by activating BamD. These results imply that BamA and BamD interact directly with OMP substrates.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Proteínas Ligadas a Lipídeos/fisiologia , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Códon/genética , Sequência Conservada , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Bicamadas Lipídicas , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Mutação de Sentido Incorreto , Mutação Puntual , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Supressão Genética , TemperaturaRESUMO
The bacterial outer membrane (OM) is an exceptional biological structure with a unique composition that contributes significantly to the resiliency of Gram-negative bacteria. Since all OM components are synthesized in the cytosol, the cell must efficiently transport OM-specific lipids and proteins across the cell envelope and stably integrate them into a growing membrane. In this review, we discuss the challenges associated with these processes and detail the elegant solutions that cells have evolved to address the topological problem of OM biogenesis. Special attention will be paid to the Bam machine, a highly conserved multiprotein complex that facilitates OM ß-barrel folding. This article is part of a Special Issue entitled: Protein Folding in Membranes.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Modelos MolecularesRESUMO
The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3, but also revealed how quickly viral escape can curtail effective options4,5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including Evusheld™ and its constituent, cilgavimab4,6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protection in vivo against the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.
RESUMO
Biogenesis of the outer membrane (OM) is an essential process in gram-negative bacteria. One of the key steps of OM biogenesis is the assembly of integral outer membrane beta-barrel proteins (OMPs) by a protein machine called the Bam complex. In Escherichia coli, the Bam complex is composed of the essential proteins BamA and BamD and three nonessential lipoproteins, BamB, BamC, and BamE. Both BamC and BamE are important for stabilizing the interaction between BamA and BamD. We used comprehensive genetic analysis to clarify the interplay between BamA and the BamCDE subcomplex. Combining a ΔbamE allele with mutations in genes that encode other OMP assembly factors leads to severe synthetic phenotypes, suggesting a critical function for BamE. These synthetic phenotypes are not nearly as severe in a ΔbamC background, suggesting that the functions of BamC and BamE are not completely overlapping. This unique function of BamE is related to the conformational state of BamA. In wild-type cells, BamA is sensitive to externally added proteinase K. Strikingly, when ΔbamE mutant cells are treated with proteinase K, BamA is degraded beyond detection. Taken together, our findings suggest that BamE modulates the conformation of BamA, likely through its interactions with BamD.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Endopeptidase K/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Conformação Proteica , Mapeamento de Interação de ProteínasRESUMO
Kill switches provide a biocontainment strategy in which unwanted growth of an engineered microorganism is prevented by expression of a toxin gene. A major challenge in kill switch engineering is balancing evolutionary stability with robust cell killing activity in application relevant host strains. Understanding host-specific containment dynamics and modes of failure helps to develop potent yet stable kill switches. To guide the design of robust kill switches in the agriculturally relevant strain Pseudomonas fluorescens SBW25, we present a comparison of lethality, stability, and genetic escape of eight different toxic effectors in the presence of their cognate inactivators (i.e., toxin-antitoxin modules, polymorphic exotoxin-immunity systems, restriction endonuclease-methyltransferase pair). We find that cell killing capacity and evolutionary stability are inversely correlated and dependent on the level of protection provided by the inactivator gene. Decreasing the proteolytic stability of the inactivator protein can increase cell killing capacity, but at the cost of long-term circuit stability. By comparing toxins within the same genetic context, we determine that modes of genetic escape increase with circuit complexity and are driven by toxin activity, the protective capacity of the inactivator, and the presence of mutation-prone sequences within the circuit. Collectively, the results of our study reveal that circuit complexity, toxin choice, inactivator stability, and DNA sequence design are powerful drivers of kill switch stability and valuable targets for optimization of biocontainment systems.
Assuntos
Antitoxinas , Pseudomonas fluorescens , Pseudomonas fluorescens/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Like all outer membrane (OM) constituents, integral OM ß-barrel proteins in Gram-negative bacteria are synthesized in the cytoplasm and trafficked to the OM, where they are locally assembled into the growing OM by the ubiquitous ß-barrel assembly machine (Bam). While the identities and structures of all essential and accessory Bam components have been determined, the basic mechanism of Bam-assisted OM protein integration remains elusive. Here we review mechanistic analyses of OM ß-barrel protein folding and Bam dynamics and summarize recent insights that inform a general model for OM protein recognition and assembly by the Bam complex.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bactérias Gram-Negativas/química , Dobramento de Proteína , Escherichia coli/química , Proteínas de Escherichia coli/química , Transporte ProteicoRESUMO
Bitter taste perception in humans is mediated by receptors encoded by 25 genes that together comprise the TAS2R (or T2R) gene family. The ability to identify the ligand(s) for each of these receptors is dependent on understanding allelic variation in TAS2R genes, which may have a significant effect on ligand recognition. To investigate the extent of coding variation among TAS2R alleles, we performed a comprehensive evaluation of sequence and haplotype variation in the human bitter taste receptor gene repertoire. We found that these genes exhibit substantial coding sequence diversity. In a worldwide population sample of 55 individuals, we found an average of 4.2 variant amino acid positions per gene. In aggregate, the 24 genes analyzed here, along with the phenylthiocarbamide (PTC) receptor gene analyzed previously, specify 151 different protein coding haplotypes. Analyses of the ratio of synonymous and nonsynonymous nucleotide substitutions using the Ka/Ks ratio revealed an excess of amino acid substitutions relative to most other genes examined to date (Ka/Ks = 0.94). In addition, comparisons with more than 1,500 other genes revealed that levels of diversity in the TAS2R genes were significantly greater than expected (pi = 0.11%; p < 0.01), as were levels of differentiation among continental populations (FST = 0.22; p < 0.05). These diversity patterns indicate that unusually high levels of allelic variation are found within TAS2R loci and that human populations differ appreciably with respect to TAS2R allele frequencies. Diversity in the TAS2R genes may be accounted for by natural selection, which may have favored alleles responsive to toxic, bitter compounds found in plants. These findings are consistent with the view that different alleles of the TAS2R genes encode receptors that recognize different ligands, and suggest that the haplotypes we have identified will be important in studies of receptor-ligand recognition.
Assuntos
Variação Genética , Haplótipos/genética , Receptores de Superfície Celular/genética , Paladar , Alelos , Humanos , Ligantes , Polimorfismo de Nucleotídeo Único , Receptores Acoplados a Proteínas G , Seleção Genética , Análise de Sequência de DNA , Papilas Gustativas/fisiologiaRESUMO
The central Bam components BamA and BamD are both essential genes in E. coli, a fact that often confounds genetic analysis using classical methods. The isolation of "depletion strains" in which these genes can be conditionally expressed removes this obstacle and facilitates the in vivo characterization of Bam function. This chapter describes an efficient two-step recombineering method for the construction of such a depletion strain, which contains an arabinose-inducible allele of bamD, using the λ Red system. Additionally, a simple protocol is presented for the depletion of bamD expression in live cells, which is particularly useful for the characterization of mutant alleles of bamD (complementation analysis). In principle, the procedures described can be adapted to produce and characterize depletion strains for any essential gene in E. coli or any other bacterium that is similarly amenable to genome engineering.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Engenharia Genética/métodos , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Recombinação GenéticaRESUMO
UNLABELLED: The seven human sirtuins are a family of ubiquitously expressed and evolutionarily conserved NAD(+)-dependent deacylases/mono-ADP ribosyltransferases that regulate numerous cellular and organismal functions, including metabolism, cell cycle, and longevity. Here, we report the discovery that all seven sirtuins have broad-range antiviral properties. We demonstrate that small interfering RNA (siRNA)-mediated knockdown of individual sirtuins and drug-mediated inhibition of sirtuin enzymatic activity increase the production of virus progeny in infected human cells. This impact on virus growth is observed for both DNA and RNA viruses. Importantly, sirtuin-activating drugs inhibit the replication of diverse viruses, as we demonstrate for human cytomegalovirus, a slowly replicating DNA virus, and influenza A (H1N1) virus, an RNA virus that multiplies rapidly. Furthermore, sirtuin defense functions are evolutionarily conserved, since CobB, the sirtuin homologue in Escherichia coli, protects against bacteriophages. Altogether, our findings establish sirtuins as broad-spectrum and evolutionarily conserved components of the immune defense system, providing a framework for elucidating a new set of host cell defense mechanisms and developing sirtuin modulators with antiviral activity. IMPORTANCE: We live in a sea of viruses, some of which are human pathogens. These pathogenic viruses exhibit numerous differences: DNA or RNA genomes, enveloped or naked virions, nuclear or cytoplasmic replication, diverse disease symptoms, etc. Most antiviral drugs target specific viral proteins. Consequently, they often work for only one virus, and their efficacy can be compromised by the rapid evolution of resistant variants. There is a need for the identification of host proteins with broad-spectrum antiviral functions, which provide effective targets for therapeutic treatments that limit the evolution of viral resistance. Here, we report that sirtuins present such an opportunity for the development of broad-spectrum antiviral treatments, since our findings highlight these enzymes as ancient defense factors that protect against a variety of viral pathogens.