mBio最新文献

Cell division is a fundamental process ensuring the perpetuation of all cellular life forms. Archaea of the order Sulfolobales divide using a simpler version of the eukaryotic endosomal sorting complexes required for transport (ESCRT) machinery, composed of three ESCRT-III homologs (ESCRT-III, -III-1, and -III-2), AAA+ ATPase Vps4 and an archaea-specific component CdvA. Here, we clarify how these components act sequentially to drive the division of the hyperthermophilic archaeon Saccharolobus islandicus. Our data suggest that ESCRT-III plays an active role during the early stage of membrane constriction during cytokinesis, whereas ESCRT-III-1 and ESCRT-III-2 are indispensable for the "pre-late" and "late" stages of cytokinesis, respectively. In the escrt-III-1 deletion strain, the division is blocked when the mid-cell constriction reaches ~30% of the initial cell diameter ("pre-late" stage), yielding "chain-like" cellular aggregates. Depletion of ESCRT-III-2 leads to the accumulation of cells connected through narrow membrane bridges ("late" stage), consistent with the key role of this protein in the final membrane abscission. We used 3D-single molecule localization microscopy to image ESCRT-III rings of different diameters and show that the decrease in the ESCRT-III ring diameter and membrane constriction are inconsistent with a mechanism exclusively based on spiraling of the ESCRT-III filaments. By contrast, the cone-shaped assemblies of ESCRT-III-1 and ESCRT-III-2 are consistent with spiral formation, highlighting the distinct roles of the three ESCRT-III proteins during the cytokinesis. We propose the "relay race" model, whereby the cytokinesis is achieved through a sequential and concerted action of different ESCRT machinery components.

Importance: Two major cytokinesis mechanisms, rooted in contractile FtsZ and endosomal sorting complexes required for transport (ESCRT) rings, respectively, have emerged in the course of evolution. Whereas bacteria rely on the FtsZ-based mechanism, different lineages of archaea use either of the two systems, and eukaryotes have inherited the ESCRT-based cell division machinery from their archaeal ancestors. The mechanism of ESCRT-based cell division in archaea remains poorly understood and mechanistic studies on different archaeal model systems are essential to unravel the natural history of the ESCRT machinery. Here we investigate the interplay between three major ESCRT-III homologs during the division of a hyperthermophilic archaeon Saccharolobus islandicus and propose the "relay race" model of cytokinesis.

High-throughput DNA transformation techniques are invaluable when generating high-diversity mutant libraries, a cornerstone of successful protein engineering. However, transformation efficiencies have a direct correlation with the probability of introducing multiple DNA molecules into each cell, although reliable library screenings require cells that contain a single unique genotype. Thus, transformation methods that yield a high multiplicity of transformations are unsuitable for high-diversity library screenings. Here, we describe an innovative yeast library transformation method that is both simple and highly efficient. Our dual heat-shock and electroporation approach (HEEL) creates high-quality DNA libraries by increasing the fraction of mono-transformed yeast cells from 20% to over 70% of all transformed cells, thus allowing for near-perfect phenotype-to-genotype associations. HEEL also allows more than 10⁷ yeast cells per reaction to be transformed with a circular plasmid molecule, which corresponds to an almost 100-fold improvement compared with current yeast transformation methods. To further refine our library screening approach, we integrated an automated yeast genotyping workflow with a dual-barcode design that employs both a single nucleotide polymorphism and a high-diversity region. This design allows for robust identification and quantification of unique genotypes within a heterogeneous population using standard Sanger sequencing. Our findings demonstrate that the longstanding trade-off between the size and quality of transformed yeast libraries can be overcome. By employing the HEEL method, large DNA libraries can be transformed into yeast with high-efficiency, while maintaining high library quality, essential for successful mutant screenings. This advancement holds significant promise for the fields of molecular biology and protein engineering.IMPORTANCEWith the recent expansion of artificial intelligence in the field of synthetic biology, there has never been a greater need for high-quality data and reliable measurements of phenotype-to-genotype relationships. However, one major obstacle to creating accurate computer-based models is the current abundance of low-quality phenotypic measurements originating from numerous high-throughput but low-resolution assays. Rather than increasing the quantity of measurements, new studies should aim to generate as accurate measurements as possible. The HEEL methodology presented here aims to address this issue by minimizing the problem of multi-plasmid uptake during high-throughput yeast DNA transformations, which leads to the creation of heterogeneous cellular genotypes. HEEL should enable highly accurate phenotype-to-genotype measurements going forward, which could be used to construct better computer-based models.

A challenge in viral vaccine development is to produce vaccines that generate both neutralizing antibodies to prevent infection and cytotoxic CD8⁺ T-cells that target conserved viral proteins and can eliminate infected cells to control virus spread. mRNA technology offers an opportunity to design vaccines based on conserved CD8-targeting epitopes, but achieving robust antigen-specific CD8⁺ T-cells remains a challenge. Here, we tested the viral-derived oligonucleotide DDO268 as an adjuvant in the context of a model influenza A virus (IAV) nucleoprotein (NP) mRNA vaccine in C57BL/6 mice. DDO268 when co-packaged with mRNA in lipid nanoparticles is sensed by RIG I-like receptors and safely induces local type I interferon (IFN) production followed by dendritic cells type 1 activation and migration to the draining lymph nodes. This early response triggered by DDO268 improved the generation of IgG2c antibodies and antigen-specific Th1 CD4⁺ and CD8⁺ T-cells (IFNγ⁺TNFα⁺IL2⁺) that provided enhanced protection against lethal IAV challenge. In addition, the inclusion of DDO268 reduced the antigen dose required to achieve protection. These results highlight the potential of DDO268 as an effective mRNA vaccine adjuvant and show that an IAV NP mRNA/DDO268 vaccine is a promising approach for generating protective immunity against conserved internal IAV epitopes.IMPORTANCEVaccines that generate neutralizing antibodies and cytotoxic CD8⁺ T-cells targeting conserved epitopes are ideal for effective protection against viruses. mRNA vaccines combined with the right adjuvant offer a promising solution to this challenge. We show that the virus-derived oligonucleotide DDO268 enhances antibody and T-cell responses to an influenza A virus (IAV) nucleoprotein mRNA vaccine in mice. DDO268 safely induces local type I interferon production and stimulates dendritic cell activation providing enhanced protection against IAV challenge. In addition, the adjuvant activity of DDO268 allows for the use of lower antigen doses during vaccination.

Unique for a eukaryote, protein-coding genes in trypanosomes are arranged in polycistronic transcription units (PTUs). This genome arrangement has led to a model where Pol II transcription of PTUs is unregulated and changes in gene expression are entirely post-transcriptional. Trypanosoma brucei brucei is unable to infect humans because of its susceptibility to an innate immune complex, trypanosome lytic factor (TLF) in the circulation of humans. The initial step in TLF-mediated lysis of T.b.brucei requires high affinity haptoglobin/hemoglobin receptor (HpHbR) binding. Here, we demonstrate that by in vitro selection with TLF, resistance is obtained in a stepwise process correlating with loss of HpHbR expression at an allelic level. RNA-seq, Pol II ChIP, and run-on analysis indicate HpHbR silencing is at the transcriptional level, where loss of Pol II binding at the promoter region specifically shuts down transcription of the HpHbR-containing gene cluster and the adjacent opposing gene cluster. Reversible transcriptional silencing of the divergent PTUs correlates with DNA base J modification of the shared promoter region. Base J function in establishing transcriptional silencing, rather than maintenance, is suggested by the maintenance of PTU silencing following the inhibition of J-biosynthesis and subsequent loss of the modified DNA base. Therefore, we show that epigenetic mechanisms exist to regulate gene expression via Pol II transcription initiation of gene clusters in a mono-allelic fashion. These findings suggest epigenetic chromatin-based regulation of gene expression is deeply conserved among eukaryotes, including early divergent eukaryotes that rely on polycistronic transcription.IMPORTANCEThe single-cell parasite Trypanosoma brucei causes lethal diseases in both humans and livestock. T. brucei undergoes multiple developmental changes to adapt in different environments during its digenetic life cycle. With protein-coding genes organized as polycistronic transcription and apparent absence of promoter-mediated regulation of transcription initiation, it is believed that developmental gene regulation in trypanosomes is essentially post-transcriptional. In this study, we found reversible Pol II transcriptional silencing of two adjacent polycistronic gene arrays that correlate with the novel DNA base J modification of the shared promoter region. Our findings support epigenetic regulation of Pol II transcription initiation as a viable mechanism of gene expression control in T. brucei. This has implications for our understanding how trypanosomes utilize polycistronic genome organization to regulate gene expression during its life cycle.

The bacterium Pseudomonas aeruginosa is an opportunistic pathogen that can cause lung, skin, wound, joint, urinary tract, and eye infections. While P. aeruginosa is known to exhibit a robust competitive response toward other bacterial species, this bacterium is frequently identified in polymicrobial infections where multiple species survive. For example, in prosthetic joint infections, P. aeruginosa can be identified along with other pathogenic bacteria including Staphylococcus aureus, Enterococcus faecalis, and Corynebacterium striatum. Here, we have explored the survival and behavior of such microbes and find that E. faecalis readily survives culturing with P. aeruginosa while other tested species do not. In each of the tested conditions, E. faecalis growth remained unchanged by the presence of P. aeruginosa, indicating a unique mutualistic interaction between the two species. We find that E. faecalis proximity leads P. aeruginosa to attenuate competitive behaviors as exemplified by reduced production of Pseudomonas quinolone signal and pyocyanin. Reduced alkyl quinolones are important to E. faecalis as these will grow in supernatant from a quinolone mutant but not P. aeruginosa wild-type in planktonic culture. The reduced pyocyanin production of P. aeruginosa is attributable to production of ornithine by E. faecalis, which we recapitulate by adding exogenous ornithine to P. aeruginosa monocultures. Similarly, co-culture with an ornithine-deficient strain of E. faecalis leads P. aeruginosa to yield near monoculture amounts of pyocyanin. Here, we directly demonstrate how notorious pathogens such as P. aeruginosa might persist in polymicrobial infections under the influence of metabolites produced by other bacterial species.

Importance: While we now appreciate that many infections are polymicrobial, we understand little of the specific actions between a given set of microbes to enable combinatorial survival and pathogenesis. The bacteria Pseudomonas aeruginosa and Enterococcus faecalis are both prevalent pathogens in wound, urinary tract, and bacteremic infections. While P. aeruginosa often kills other species in standard laboratory culture conditions, we present here that E. faecalis can be reliably co-cultured with P. aeruginosa. We specifically detail that ornithine produced by E. faecalis reduces the Pseudomonas quinolone signal response of P. aeruginosa. This reduction of the Pseudomonas quinolone signal response aids E. faecalis growth.

HIV-1 unspliced RNA serves two distinct functions during viral replication: it is packaged into particles as the viral genome, and it is translated to generate Gag/Gag-Pol polyproteins required for virus assembly. Recent studies have demonstrated that in cultured cells, HIV-1 uses multiple transcription start sites to generate several unspliced RNA species, including two major transcripts with three and one 5' guanosine, referred to as 3G and 1G RNA, respectively. Although nearly identical, 1G RNA is selected over 3G RNA to be packaged as the virion genome, indicating that these RNA species are functionally distinct. Currently, our understanding of HIV-1 transcription start site usage and the functions of RNA species is based on studies using cultured cells. Here, we examined samples from people living with HIV to investigate HIV-1 transcription start site usage and its impact on RNA function. Using paired samples collected from the same participants on the same date, we examined the HIV-1 unspliced RNA species in infected cells (PBMC) and in viruses (plasma). Our findings demonstrate that in people living with HIV, the virus uses multiple transcription start sites to generate several unspliced transcripts, including 3G and 1G RNA. Furthermore, we observed an enrichment of 1G RNA in the paired plasma samples, indicating a preferential packaging of 1G RNA in vivo. Our study illustrates the complex regulation of HIV-1 unspliced RNA in people living with HIV and provides valuable insights into how HIV-1 unspliced RNAs serve their functions in vivo.IMPORTANCEHIV-1 virions must contain unspliced RNA and its translation products to maintain infectivity. How HIV-1 unspliced RNA fulfills these two essential and yet distinct roles in viral replication has been a long-standing question in the field. In this report, we demonstrate that in people living with HIV, the virus uses multiple transcription start sites to generate several unspliced RNA species that are 99% identical in sequence but differ functionally. One of the RNA species, 1G RNA, is selected over other HIV-1 unspliced RNAs to be packaged into viral particles. These findings are consistent with previous cell-culture-based observations and provide insights into how HIV-1 regulates its unspliced RNA function in people living with HIV.

Respiratory epithelial cells can survive direct infection by influenza viruses, and the long-term consequences of that infection have been characterized in a subset of proximal airway cell types. The impact on the cells that survive viral infection in the distal lung epithelia, however, is much less well-characterized. Utilizing a Cre-expressing influenza B virus (IBV) and a lox-stop-lox tdTomato reporter mouse model, we identified that alveolar type 2 (AT2) pneumocytes, a progenitor cell type in the distal lung, can survive viral infection. We show that survival of infection is associated with transcriptional dysregulation compared to bystander AT2 pneumocytes from the same lung. Furthermore, ex vivo experiments revealed a significant reduction in proliferation rates in survivor AT2 pneumocytes compared to matched, non-directly infected bystander cells. Our findings not only enhance our understanding of the AT2 pneumocyte response to IBV infection but could also have broader implications for the mechanisms of respiratory epithelial repair post-viral infection.

Importance: Alveolar type 2 (AT2) pneumocytes are a cell type critical for repair of the distal lung after an injury, such as a viral infection. After epithelial damage, AT2 pneumocytes proliferate for both self-renewal and differentiation into type I pneumocytes to repopulate the epithelium. Theoretically, some of the long-term lung sequelae associated with viral infections could be the result of inappropriate AT2 behavior. Here, the authors report that during an influenza B virus infection, some of the actively infected AT2 pneumocytes can ultimately eliminate all traces of the viral RNA and persist in the host long term. As a consequence of having been infected, however, the cells display an altered transcriptional profile and decreased proliferative capacity. These data together suggest a mechanism for how an acute viral infection can have long-term impacts on the pulmonary system.

Cryptococcus neoformans is a fungal pathogen that can cause lethal disease in immunocompromised patients. Immunocompetent host immune responses, such as formation of pulmonary granulomas, control the infection and prevent disseminated disease. Little is known about the immunological conditions establishing the latent infection granuloma in the lungs. To investigate this, we performed an analysis of pulmonary immune cell populations, cytokine changes, and granuloma formation during infection with a latent disease-causing clinical isolate in C3HeB/FeJ mice over 360 days. We found that latently infected mice progress through three phases of granuloma formation where different immune profiles dominate: an early phase characterized by eosinophilia, high IL-4/IL-13, and C. neoformans proliferation in the lungs; an intermediate phase characterized by multinucleated giant cell formation, high IL-1α/IFNγ, granuloma expansion, and increased blood antigen levels; and a late phase characterized by a significant expansion of T cells, granuloma condensation, and decreases in lung fungal burden and blood antigen levels. These findings highlight a complex series of immune changes that occur during the establishment of granulomas that control C. neoformans in the lungs and lay the foundation for studies to identify critical beneficial immune responses to Cryptococcus infections.IMPORTANCECryptococcus neoformans is a fungal pathogen that disseminates from the lungs to the brain to cause fatal disease. Latent C. neoformans infection in the lungs is controlled by organized collections of immune cells called granulomas. The formation and structure of Cryptococcus granulomas are poorly understood due to inconsistent human pathology results and disagreement between necrotic granuloma-forming rat models and non-necrotic granuloma-forming mouse models. To overcome this, we investigated granuloma formation during latent C. neoformans infection in the C3HeB/FeJ mouse strain which forms necrotic lung granulomas in response to other pathogens. We found that latent C. neoformans granuloma formation progresses through phases that we described as early, intermediate, and late with different immune response profiles and granulomatous characteristics. Ultimately, we show that C3HeB/FeJ mice latently infected with C. neoformans form non-necrotic granulomas and could provide a novel mouse model to investigate host immune response profiles.

Fusobacterium is a bacterium associated with colorectal cancer (CRC) tumorigenesis, progression, and metastasis. Fap2 is a fusobacteria-specific outer membrane galactose-binding lectin that mediates Fusobacterium adherence to and invasion of CRC tumors. Advances in omics analyses provide an opportunity to profile and identify microbial genomic features that correlate with the cancer-associated bacterial virulence factor Fap2. Here, we analyze genomes of Fusobacterium colon tumor isolates and find that a family of post-translational modification enzymes containing Fic domains is associated with Fap2 positivity in these strains. We demonstrate that Fic family genes expand with the presence of Fap2 in the fusobacterial pangenome. Through comparative genomic analysis, we find that Fap2⁺ Fusobacteriota are highly enriched with Fic gene families compared to other cancer-associated and human gut microbiome bacterial taxa. Using a global data set of CRC shotgun metagenomes, we show that fusobacterial Fic and Fap2 genes frequently co-occur in the fecal microbiomes of individuals with late-stage CRC. We further characterize specific Fic gene families harbored by Fap2⁺ Fusobacterium animalis genomes and detect recombination events and elements of horizontal gene transfer via synteny analysis of Fic gene loci. Exposure of a F. animalis strain to a colon adenocarcinoma cell line increases gene expression of fusobacterial Fic and virulence-associated adhesins. Finally, we demonstrate that Fic proteins are synthesized by F. animalis as Fic peptides are detectable in F. animalis monoculture supernatants. Taken together, our study uncovers Fic genes as potential virulence factors in Fap2⁺ fusobacterial genomes.IMPORTANCEAccumulating data support that bacterial members of the intra-tumoral microbiota critically influence colorectal cancer progression. Yet, relatively little is known about non-adhesin fusobacterial virulence factors that may influence carcinogenesis. Our genomic analysis and expression assays in fusobacteria identify Fic domain-containing genes, well-studied virulence factors in pathogenic bacteria, as potential fusobacterial virulence features. The Fic family proteins that we find are encoded by fusobacteria and expressed by Fusobacterium animalis merit future investigation to assess their roles in colorectal cancer development and progression.