Single Gene Deletion Research Articles

Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V-F CRISPR/Cas Nuclease AsCas12f1.

Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces. However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.

Implications of two-component systems EnvZ/OmpR and BaeS/BaeR in in vitro temocillin resistance in Escherichia coli.

BaeS/BaeR is a two-component system of Escherichia coli that controls the expression of porins and efflux pumps. Its role in beta-lactam resistance is limited. To study the role of baeS/baeR two-component system in temocillin resistance in E. coli. E. coli strain BW25113 and single-gene deletion mutants related to two-component systems were collected from the KEIO collection. Double-gen deletion mutants were generated. Temocillin-resistant mutant frequencies were determined at 32 mg/L. E. coli BW25113 mutants were selected by selective pressure from serial passages. Biological costs were analysed by growth curves. Genomes of the generated mutants were sequenced. The expression level of the mdtA, mdtB, mdtC, acrD and tolC in the ΔbaeS mutant was determined by RT-PCR (with/without temocillin exposure). The frequency of temocillin mutants ranged from 2.12 × 10-8 to 4.51 × 10-8 in single-porin mutants. No mutants were recovered from E. coli BW25113 (>10-9). Selection of temocillin-resistant variants by serial passage yielded mutants up to 128 mg/L. Mutations were found in the baeS gene. Temocillin MICs ranged from 4 to 32 mg/L (highest MICs for ΔbaeS and ΔompR). The efflux pumps mdtA, mdtB, mdtC and acrD pumps were overexpressed 3-10-fold in the presence of temocillin in ΔbaeS compared to control. Mutations in the sensor histidine kinase, baeS, may be involved in temocillin resistance through the expression of the efflux pumps mdtABC and acrD. In addition, the low mutation rate may be a good predictor of temocillin activity.

Identification of L11L and L7L as virulence-related genes in the African swine fever virus genome.

African swine fever (ASF) is an infectious disease that causes considerable economic losses in pig farming. The agent of this disease, African swine fever virus (ASFV), is a double-stranded DNA virus with a capsid membrane and a genome that is 170-194 kb in length encoding over 150 proteins. In recent years, several live attenuated strains of ASFV have been studied as vaccine candidates, including the SY18ΔL7-11. This strain features deletion of L7L, L8L, L9R, L10L and L11L genes and was found to exhibit significantly reduced pathogenicity in pigs, suggesting that these five genes play key roles in virulence. Here, we constructed and evaluated the virulence of ASFV mutations with SY18ΔL7, SY18ΔL8, SY18ΔL9, SY18ΔL10, and SY18ΔL11L. Our findings did not reveal any significant differences in replication efficiency between the single-gene deletion strains and the parental strains. Pigs inoculated with SY18ΔL8L, SY18ΔL9R and SY18ΔL10L exhibited clinical signs similar to those inoculated with the parental strains. Survival rate of pigs inoculated with 103.0TCID50 of SY18ΔL7L was 25%, while all pigs inoculated with 103.0TCID50 of SY18ΔL11L survived, and 50% inoculated with 106.0TCID50 SY18ΔL11L survived. The results indicate that L8L, L9R and L10L do not affect ASFV SY18 virulence, while the L7L and L11L are associated with virulence.

Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP

BackgroundClostridium sp. AWRP (AWRP) is a novel acetogenic bacterium isolated under high partial pressure of carbon monoxide (CO) and can be one of promising candidates for alcohol production from carbon oxides. Compared to model strains such as C. ljungdahlii and C. autoethanogenum, however, genetic manipulation of AWRP has not been established, preventing studies on its physiological characteristics and metabolic engineering.ResultsWe were able to demonstrate the genetic domestication of AWRP, including transformation of shuttle plasmids, promoter characterization, and genome editing. From the conjugation experiment with E. coli S17-1, among the four replicons tested (pCB102, pAMβ1, pIP404, and pIM13), three replicated in AWRP but pCB102 was the only one that could be transferred by electroporation. DNA methylation in E. coli significantly influenced transformation efficiencies in AWRP: the highest transformation efficiencies (102–103 CFU/µg) were achieved with unmethylated plasmid DNA. Determination of strengths of several clostridial promoters enabled the establishment of a CRISPR/Cas12a genome editing system based on Acidaminococcus sp. BV3L6 cas12a gene; interestingly, the commonly used CRISPR/Cas9 system did not work in AWRP, although it expressed the weakest promoter (C. acetobutylicum Pptb) tested. This system was successfully employed for the single gene deletion (xylB and pyrE) and double deletion of two prophage gene clusters.ConclusionsThe presented genome editing system allowed us to achieve several genome manipulations, including double deletion of two large prophage groups. The genetic toolbox developed in this study will offer a chance for deeper studies on Clostridium sp. AWRP for syngas fermentation and carbon dioxide (CO2) sequestration.

Limited threat of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletion to the utility of HRP2-based malaria RDTs in Northern Uganda

BackgroundRapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein-2 (PfHRP2) are exclusively deployed in Uganda, but deletion of the pfhrp2/3 target gene threatens their usefulness as malaria diagnosis and surveillance tools.MethodsA cross-sectional survey was conducted at 40 sites across four regions of Uganda in Acholi, Lango, W. Nile and Karamoja from March 2021 to June 2023. Symptomatic malaria suspected patients were recruited and screened with both HRP2 and pan lactate dehydrogenase (pLDH) detecting RDTs. Dried blood spots (DBS) were collected from all patients and a random subset were used for genomic analysis to confirm parasite species and pfhrp2 and pfhrp3 gene status. Plasmodium species was determined using a conventional multiplex PCR while pfhrp2 and pfhrp3 gene deletions were determined using a real-time multiplex qPCR. Expression of the HRP2 protein antigen in a subset of samples was further assessed using a ELISA.ResultsOut of 2435 symptomatic patients tested for malaria, 1504 (61.8%) were positive on pLDH RDT. Overall, qPCR confirmed single pfhrp2 gene deletion in 1 out of 416 (0.2%) randomly selected samples that were confirmed of P. falciparum mono-infections.ConclusionThese findings show limited threat of pfhrp2/3 gene deletions in the survey areas suggesting that HRP2 RDTs are still useful diagnostic tools for surveillance and diagnosis of P. falciparum malaria infections in symptomatic patients in this setting. Periodic genomic surveillance is warranted to monitor the frequency and trend of gene deletions and its effect on RDTs.

Development of a lambda Red based system for gene deletion in Chlamydia.

The ability to efficiently target loci in the Chlamydia trachomatis genome for deletion remains a desirable goal in the field and new strategies need to be developed and refined. Here we describe the development and application of a lambda red recombineering system for Chlamydia. Using a non-replicative plasmid encoding key lambda Red components and targeting sequences, we demonstrate the efficient deletion of numerous gene targets in the model chlamydial strains C. trachomatis L2/434 and C. muridarum. For initial development of the system, we targeted the incA gene of C. trachomatis for deletion. Deletion mutants containing a chloramphenicol resistance marker were recovered within 3 rounds of selection, or 2 rounds of passaging, in McCoy cells and the resulting clones (CTΔincA) were verified by PCR-based genotyping and whole genome sequencing. Phenotypic assessment of host cells infected with CTΔincA was performed by immunofluorescence microscopy and confirmed the lack of IncA expression and the uniform presence of nonfusogenic vacuoles (inclusions) across CTΔincA-infected monolayers. To explore the utility of this system, we deleted 5 additional candidate virulence factors in C. trachomatis and C. muridarum, including deletions of single and multiple genes. We expect lambda Red recombineering to offer a powerful new strategy for making gene deletion and/or replacement mutants in Chlamydia.

Screening of genes involved in phage-resistance of Escherichia coli and effects of substances interacting with primosomal protein A on the resistant bacteria.

The study was to identify the genes involved in phage resistance and to develop an effective biocontrol method to improve the lytic activity of phages against foodborne pathogens. A total of 3,909 single gene-deletion mutants of Escherichia coli BW25113 from the Keio collection were individually screened for genes involved in phage resistance. Phage S127BCL3 isolated from chicken liver, infecting both E. coli BW25113 and O157: H7, was characterized and used for screening. The 10 gene-deletion mutants showed increased susceptibility to phage S127BCL3. Among them, priA gene-deletion mutant strain showed significant susceptibility to the phages S127BCL3 and T7. Furthermore, we investigated the substances that have been reported to inhibit the function of primosomal protein A (PriA) and were used to confirm increased phage susceptibility in E. coli BW25113 (Parent strain) and O157: H7. PriA inhibitors at a low concentration showed combined effects with phage against E. coli O157: H7 and delayed the regrowth rate of phage-resistant cells.

A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas.

Gene knockout is a widely used technique for engineering bacterial genomes, investigating the roles of genes in metabolism, and conferring biological characteristics. Herein, we developed a rapid, efficient, and simple method for the knockout of long gene cassettes in Pseudomonas spp., based on a traditional allelic exchange strategy. The upstream and downstream sequences of the target gene cluster to be deleted were amplified using primers with 5'-end sequences identical to the multiple cloning sites of a suicide plasmid (mutant allele insert vector). The sequences were then fused with the linearized suicide plasmid in one step via seamless cloning. The resulting allelic exchange vector (recombinant plasmid) was introduced from the donor strain (Escherichia coli SM 10) into recipient cells (Pseudomonas putida, P. composti, and P. khazarica) via conjugation. Single-crossover merodiploids (integrates the vector into host chromosome by homologous recombination) were screened based on antibiotic resistance conferred by the plasmid, and double-crossover haploids (deleting the target gene clusters and inserted alien plasmid backbone) were selected using sucrose-mediated counterselection. Unlike other approaches, the method described herein introduces no selective marker genes into the genomes of the knockout mutants. Using our method, we successfully deleted polysaccharide-encoding gene clusters in P. putida, P. composti, and P. khazarica and generated four mutants with single-gene cassette deletions up to 18 kbp and one mutant with double-gene cassette deletion of approximately 34 kbp. Collectively, our results indicate that this method is ideal for the deletion of long genetic sequences, yielding seamless mutants of various Pseudomonas spp.

Compound Loss of Dnmt3a and Kmt2c in Myeloid Malignancies

Monosomy 7, or loss of 7q (-7/del(7q)), is one the most common chromosomal anomalies in myelodysplastic syndromes (MDS, ~10% of patients), a malignant disorder of the blood system with few treatment options. There are currently no targeted therapies available for -7/del(7q) MDS, and patients progress to a deadly acute myeloid leukemia (AML). The 7q segment is rich in genes that are implicated in MDS pathophysiology, including KMT2C encoding a histone H3 lysine 4 methyltransferase (depositing H3K4me1) that marks active gene enhancers during myeloid differentiation. Co-occurring DNA methyltransferase DNMT3A mutations are significantly overrepresented in -7/del(7q) MDS, especially in the absence of other chromosomal aberrations (23%). Individually, loss in DNMT3A activity in hematopoietic stem cells (HSCs) has shown bias towards self-renewal due to a failure to shut off stem-cell-related genes, whereas a loss of KMT2C leads to aberrant myelopoiesis. Generally, only one allele of KMT2C and DNMT3A is lost in MDS, suggesting that these haploinsufficient tumor suppressors may need to cooperate to drive disease. We hypothesized that disruption of Dnmt3a cooperated with loss of Kmt2c creating an aberrant epigenetic state that may accelerate development of MDS and progression to AML. To address this, we generated mice that lacked one copy of both Kmt2c and Dnmt3a in the bone marrow by conditional deletion with a hematopoietic-specific inducible Mx1-driven Cre recombinase. After inducing Cre-mediated recombination by poly(I:C) treatment creating Kmt2c(+/del):Dnmt3a(+/del) animals, the hematopoietic stem and progenitor compartment was subjected to comprehensive immunophenotypic and functional characterization. In serial competitive bone marrow repopulation assays, cells with combined heterozygous loss of both Dnmt3a and Kmt2c demonstrated increased competitive advantage in secondary transplants and retained ability to reconstitute tri-lineage hematopoiesis in tertiary transplants, unlike single gene deletion and wild-type controls. Consistently, immunophenotypic analysis of the bone marrow compartment 16 weeks after the third transplant demonstrated persistence of the primitive long-term hematopoietic stem cells (LT-HSCs) defined as Lineage -Sca1 +cKit + (LSK) CD150 +CD48 -, which were absent in other genotypes. Further, we found expansion of the LT-HSCs in aged 80-week old Kmt2c(+/del):Dnmt3a(+/del) animals that were also characterized by a gain in self-renewal as seen by their ability to serially replate in semisolid media (MethoCult GM3434). To test if combined loss of Kmt2c and Dnmt3a led to disease development, next we established a large cohort of animals non-competitively transplanted with Kmt2c(+/del):Dnmt3a(+/del), single gene deletion, and wild-type control bone marrow, and initiated a tumor watch by monthly monitoring the composition of their peripheral blood by CBCs and flow cytometry. Sixty weeks after transplantation, over a half of double knock-out animals developed myeloid disease characterized by neutrophilia and/or monocytosis accompanied by anemia, occasional thrombocytopenia, and splenomegaly most consistent with myelodysplasia. Immunophenotypically, we found expanded LSK populations (LT-HSCs specifically) in the bone marrow and evidence of extramedullary hematopoiesis in the spleen. At the same time, the majority of mice with single gene deletion and all wild-type controls remained healthy. Overall, our studies demonstrate that combined loss of Kmt2c and Dnmt3a results in the development of myelodysplastic disease with long latency through endowing the HSCs with enhanced self-renewal capacity. We are currently interrogating the epigenetic profiles and gene expression patterns resulting from combined disruption of Dnmt3a and Kmt2c aiming to identify gene targets and signaling networks for future therapeutic intervention. Better understanding of the molecular pathogenesis of the -7/del(7q) MDS with concurrent DNMT3A mutations is critical to develop more effective treatment approaches and to improve outcomes for MDS patients.

Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a β-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin β-lactone warheads. Introduction

Systematic investigation of recipient cell genetic requirements reveals important surface receptors for conjugative transfer of IncI2 plasmids

Bacterial conjugation is a major horizontal gene transfer mechanism. While the functions encoded by many conjugative plasmids have been intensively studied, the contribution of recipient chromosome-encoded genes remains largely unknown. Here, we analyzed the genetic requirement of recipient cells for conjugation of IncI2 plasmid TP114, which was recently shown to transfer at high rates in the gut microbiota. We performed transfer assays with ~4,000 single-gene deletion mutants of Escherichia coli. When conjugation occurs on a solid medium, we observed that recipient genes impairing transfer rates were not associated with a specific cellular function. Conversely, transfer assays performed in broth were largely dependent on the lipopolysaccharide biosynthesis pathway. We further identified specific structures in lipopolysaccharides used as recipient cell surface receptors by PilV adhesins associated with the type IVb accessory pilus of TP114. Our strategy is applicable to study other mobile genetic elements and understand important host cell factors for their dissemination.

Two Putative Pheromone Receptors, but Not Their Cognate Pheromones, Regulate Female Fertility in the Atypical Mating Fungus Colletotrichum fructicola.

Colletotrichum fungi are a group of damaging phytopathogens with atypical mating type loci (harboring only MAT1-2-1 but not MAT1-1-1) and complex sexual behaviors. Sex pheromones and their cognate G-protein-coupled receptors are conserved regulators of fungal mating. These genes, however, lose function frequently among Colletotrichum species, indicating a possibility that pheromone signaling is dispensable for Colletotrichum sexual reproduction. We have identified two putative pheromone-receptor pairs (PPG1:PRE2, PPG2:PRE1) in C. fructicola, a species that exhibits plus-to-minus mating type switching and plus-minus-mediated mating line development. Here, we report the generation and characterization of gene-deletion mutants for all four genes in both plus and minus strain backgrounds. Single-gene deletion of pre1 or pre2 had no effect on sexual development, whereas their double deletion caused self-sterility in both the plus and minus strains. Moreover, double deletion of pre1 and pre2 caused female sterility in plus-minus outcrossing. Double deletion of pre1 and pre2, however, did not inhibit perithecial differentiation or plus-minus-mediated enhancement of perithecial differentiation. Contrary to the results with pre1 and pre2, double deletion of ppg1 and ppg2 had no effect on sexual compatibility, development, or fecundity. We concluded that pre1 and pre2 coordinately regulate C. fructicola mating by recognizing novel signal molecule(s) distinct from canonical Ascomycota pheromones. The contrasting importance between pheromone receptors and their cognate pheromones highlights the complicated nature of sex regulation in Colletotrichum fungi.

An expanded toolkit of drug resistance cassettes for Candida glabrata, Candida auris, and Candida albicans leads to new insights into the ergosterol pathway.

The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies in Candida glabrata, Candida auris, and Candida albicans have been mainly limited to the use of NatMX/SAT1 and HphMX/CaHyg for genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated that NatMX/SAT1, HphMX, KanMX, and/or BleMX drug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway of C. glabrata, C. auris, and C. albicans. Moreover, the utility of these tools has provided new insights into ERG genes and their relationship to azole resistance in Candida. Overall, we have expanded the toolkit for Candida pathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.

Identification of Streptococcus pneumoniae genes associated with hypothiocyanous acid tolerance through genome-wide screening.

Streptococcus pneumoniae is a commensal bacterium and invasive pathogen that causes millions of deaths worldwide. The pneumococcal vaccine offers limited protection, and the rise of antimicrobial resistance will make treatment increasingly challenging, emphasizing the need for new antipneumococcal strategies. One possibility is to target antioxidant defenses to render S. pneumoniae more susceptible to oxidants produced by the immune system. Human peroxidase enzymes will convert bacterial-derived hydrogen peroxide to hypothiocyanous acid (HOSCN) at sites of colonization and infection. Here, we used saturation transposon mutagenesis and deep sequencing to identify genes that enable S. pneumoniae to tolerate HOSCN. We identified 37 genes associated with S. pneumoniae HOSCN tolerance, including genes involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Single-gene deletion mutants of the identified antioxidant defense genes sodA, spxB, trxA, and ahpD were generated and their ability to survive HOSCN was assessed. With the exception of ΔahpD, all deletion mutants showed significantly greater sensitivity to HOSCN, validating the result of the genome-wide screen. The activity of hypothiocyanous acid reductase or glutathione reductase, known to be important for S. pneumoniae tolerance of HOSCN, was increased in three of the mutants, highlighting the compensatory potential of antioxidant systems. Double deletion of the gene encoding glutathione reductase and sodA sensitized the bacteria significantly more than single deletion. The HOSCN defense systems identified in this study may be viable targets for novel therapeutics against this deadly pathogen. IMPORTANCE Streptococcus pneumoniae is a human pathogen that causes pneumonia, bacteremia, and meningitis. Vaccination provides protection only against a quarter of the known S. pneumoniae serotypes, and the bacterium is rapidly becoming resistant to antibiotics. As such, new treatments are required. One strategy is to sensitize the bacteria to killing by the immune system. In this study, we performed a genome-wide screen to identify genes that help this bacterium resist oxidative stress exerted by the host at sites of colonization and infection. By identifying a number of critical pneumococcal defense mechanisms, our work provides novel targets for antimicrobial therapy.

Development of a CRISPR/Cas9D10A Nickase (nCas9)-Mediated Genome Editing Tool in Streptomyces.

Streptomycetes have a strong ability to produce a vast array of bioactive natural products (NPs) widely used in agriculture and veterinary/human medicine. The recently developed CRISPR/Cas9-based genome editing tools have greatly facilitated strain improvement for target NP overproduction as well as novel NP discovery in Streptomyces. However, CRISPR/Cas9 shows high toxicity to the host, limiting its application in many Streptomyces strains with a low DNA transformation efficiency. In this study, we developed a low-toxicity CRISPR/Cas9D10A nickase (nCas9)-based genome editing tool in the model strain Streptomyces coelicolor M145. We showed that in the presence of both targeting sgRNA and Cas proteins, utilization of nCas9 instead of Cas9 significantly reduced the toxicity to the host and greatly enhanced cell survival. Using this tool, we achieved deletion of single genes and gene clusters with efficiencies of 87-100 and 63-87%, and simultaneous deletion of two genes or gene clusters with efficiencies of 47 and 43%, respectively. The editing efficiency of nCas9 is comparable to that of the Cas9-mediated editing tool. Finally, the nCas9-based editing tool was successfully applied for genome editing in the industrial rapamycin-producing strain Streptomyces rapamycinicus, in which CRISPR/Cas9 cannot work well. We achieved the deletion of three tested genes with an efficiency of 27.2-30%. Collectively, the CRISPR/nCas9-based editing tool offers a convenient and efficient genetic modification system for the engineering of streptomycetes, particularly those with low DNA transformation efficiency.

Deletion of the non-adjacent genes UL148 and UL148D impairs human cytomegalovirus-mediated TNF receptor 2 surface upregulation.

Human cytomegalovirus (HCMV) is a prototypical β-herpesvirus which frequently causes morbidity and mortality in individuals with immature, suppressed, or senescent immunity. HCMV is sensed by various pattern recognition receptors, leading to the secretion of pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα). TNFα binds to two distinct trimeric receptors: TNF receptor (TNFR) 1 and TNFR2, which differ in regard to their expression profiles, affinities for soluble and membrane-bound TNFα, and down-stream signaling pathways. While both TNF receptors engage NFκB signaling, only the nearly ubiquitously expressed TNFR1 exhibits a death domain that mediates TRADD/FADD-dependent caspase activation. Under steady-state conditions, TNFR2 expression is mainly restricted to immune cells where it predominantly submits pro-survival, proliferation-stimulating, and immune-regulatory signals. Based on the observation that HCMV-infected cells show enhanced binding of TNFα, we explored the interplay between HCMV and TNFR2. As expected, uninfected fibroblasts did not show detectable levels of TNFR2 on the surface. Intriguingly, however, HCMV infection increased TNFR2 surface levels of fibroblasts. Using HCMV variants and BACmid-derived clones either harboring or lacking the ULb' region, an association between TNFR2 upregulation and the presence of the ULb' genome region became evident. Applying a comprehensive set of ULb' gene block and single gene deletion mutants, we observed that HCMV mutants in which the non-adjacent genes UL148 or UL148D had been deleted show an impaired ability to upregulate TNFR2, coinciding with an inverse regulation of TACE/ADAM17.

Metabolic modelling-based in silico drug target prediction identifies six novel repurposable drugs for melanoma

Despite high initial response rates to targeted kinase inhibitors, the majority of patients suffering from metastatic melanoma present with high relapse rates, demanding for alternative therapeutic options. We have previously developed a drug repurposing workflow to identify metabolic drug targets that, if depleted, inhibit the growth of cancer cells without harming healthy tissues. In the current study, we have applied a refined version of the workflow to specifically predict both, common essential genes across various cancer types, and melanoma-specific essential genes that could potentially be used as drug targets for melanoma treatment. The in silico single gene deletion step was adapted to simulate the knock-out of all targets of a drug on an objective function such as growth or energy balance. Based on publicly available, and in-house, large-scale transcriptomic data metabolic models for melanoma were reconstructed enabling the prediction of 28 candidate drugs and estimating their respective efficacy. Twelve highly efficacious drugs with low half-maximal inhibitory concentration values for the treatment of other cancers, which are not yet approved for melanoma treatment, were used for in vitro validation using melanoma cell lines. Combination of the top 4 out of 6 promising candidate drugs with BRAF or MEK inhibitors, partially showed synergistic growth inhibition compared to individual BRAF/MEK inhibition. Hence, the repurposing of drugs may enable an increase in therapeutic options e.g., for non-responders or upon acquired resistance to conventional melanoma treatments.

HDL functionality is dependent on hepatocyte stress defense factors Nrf1 and Nrf2.

High density lipoproteins (HDL) promote homeostasis and counteract stressful tissue damage that underlie cardiovascular and other diseases by mediating reverse cholesterol transport, reducing inflammation, and abrogating oxidative damage. However, metabolically stressful conditions associated with atherosclerosis can impair these effects. Hepatocytes play a major role in the genesis and maturation of circulating HDL, and liver stress elicits marked regulatory changes to circulating HDL abundance and composition, which affect its functionality. The mechanisms linking liver stress to HDL function are incompletely understood. In this study, we sought to determine whether stress defending transcription factors nuclear factor erythroid 2 related factor-1 (Nrf1) and -2 (Nrf2) promote hepatocyte production of functional HDL. Using genetically engineered mice briefly fed a mild metabolically stressful diet, we investigated the effect of hepatocyte-specific deletion of Nrf1, Nrf2, or both on circulating HDL cholesterol, protein composition, and function. Combined deletion, but not single gene deletion, reduced HDL cholesterol and apolipoprotein A1 levels as well as the capacity of HDL to accept cholesterol undergoing efflux from cultured macrophages and to counteract tumor necrosis factor α-induced inflammatory effect on cultured endothelial cells. This coincided with substantial alteration to the HDL proteome, which correlated with liver gene expression profiles of corresponding proteins. Thus, our findings show complementary actions by hepatocyte Nrf1 and Nrf2 play a role in shaping HDL abundance and composition to promote production of functionally viable HDL. Consequently, our study illuminates the possibility that enhancing stress defense programming in the liver may improve atheroprotective and perhaps other health promoting actions of HDL.

Dynamic interactome of the MHC I peptide loading complex in human dendritic cells

Dendritic cells (DCs) orchestrate immune responses by presenting antigenic peptides on major histocompatibility complex (MHC) molecules to T cells. Antigen processing and presentation via MHC I rely on the peptide-loading complex (PLC), a supramolecular machinery assembled around the transporter associated with antigen processing (TAP), which is the peptide transporter in the endoplasmic reticulum (ER) membrane. We studied antigen presentation in human DCs by isolating monocytes from blood and differentiating them into immature and mature DCs. We uncovered that during DC differentiation and maturation, additional proteins are recruited to the PLC, including B-cell receptor-associated protein 31 (BAP31), vesicle-associated membrane protein-associated protein A (VAPA), and extended synaptotagmin-1 (ESYT1). We demonstrated that these ER cargo export and contact site-tethering proteins colocalize with TAP and are within 40 nm proximity of the PLC, suggesting that the antigen processing machinery is located near ER exit- and membrane contact sites. While CRISPR/Cas9-mediated deletion of TAP and tapasin significantly reduced MHC I surface expression, single-gene deletions of the identified PLC interaction partners revealed a redundant role of BAP31, VAPA, and ESYT1 in MHC I antigen processing in DCs. These data highlight the dynamics and plasticity of PLC composition in DCs that previously was not recognized by the analysis of cell lines.

Improving therapeutic protein secretion in the probiotic yeast Saccharomyces boulardii using a multifactorial engineering approach

The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb’s innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb’s ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76–458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae’s secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.