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The emergence of genome-scale forward genetic screening techniques, such as Haploid Genetic screen and clustered regularly interspaced short palindromic repeats (CRISPR) knockout screen has opened new horizons in our understanding of virus infection biology. CRISPR screening has become a popular tool for the discovery of novel host factors for several viruses due to its specificity and efficiency in genome editing. Here, we review how CRISPR screening has revolutionized our understanding of virus-host interactions from scientific and technological viewpoints. A summary of the published screens conducted thus far to uncover virus host factors is presented, highlighting their experimental design and significant findings. We will outline relevant methods for customizing the CRISPR screening process to answer more specific hypotheses and compile a glossary of conducted CRISPR screens to show their design aspects. Furthermore, using flaviviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as examples, we hope to offer a broad-based perspective on the capabilities of CRISPR screening to serve as a reference point to guide future unbiased discovery of virus host factors.

Rab GTPases control intracellular vesicular transport, including retrograde trafficking of human papillomavirus (HPV) during cell entry, guiding the virus from the endosome to the trans-Golgi network (TGN), the Golgi apparatus, and eventually the nucleus. Rab proteins have been identified that act prior to the arrival of HPV at the TGN, but Rab proteins operating in later stages of entry remain elusive. Here, we report that knockdown of Rab6a impairs HPV entry by preventing HPV exit from the TGN and impeding intra-Golgi transport of the incoming virus. Rab6a supports HPV trafficking by facilitating the association of HPV with dynein, a motor protein complex, and BICD2, a dynein adaptor, in the TGN. L2 can bind directly to GTP-Rab6a in vitro, and excess of either GTP-Rab6a or GDP-Rab6 inhibits HPV entry, suggesting that cycling between GDP-Rab6 and GTP-Rab6 is critical. Notably, Rab6a is crucial for HPV-BICD2 and HPV-dynein association in the TGN of infected cells but not in the endosome. Our findings reveal important features of the molecular basis of HPV infection, including the discovery that HPV uses different mechanisms to engage dynein at different times during entry, and identify potential targets for therapeutic approaches to inhibit HPV infection.

Importance: Human papillomaviruses (HPVs) are small, non-enveloped DNA viruses that cause approximately 5% of human cancer. Like most other DNA viruses, HPV traffics to the nucleus during virus entry to successfully infect cells. We show here that HPV utilizes a cellular enzyme, Rab6a, during virus entry to engage the dynein molecular motor for transport along microtubules. Rab6a is required for complex formation between the HPV L2 capsid protein, dynein, and the dynein adaptor BICD2 in the trans-Golgi network (TGN). This complex is required for transport of the incoming virus out of the TGN as it journeys to the nucleus. Our findings identify potential targets for therapeutic approaches.

Iron (Fe) is a trace nutrient required by nearly all organisms. As a result of the demand for Fe and the toxicity of non-chelated cytosolic ionic Fe, regulatory systems have evolved to tightly balance Fe acquisition and usage while limiting overload. In most bacteria, including the mammalian pathogen Staphylococcus aureus, the ferric uptake regulator (Fur) is the primary transcriptional regulator controlling the transcription of genes that code for Fe uptake and utilization proteins. Fpa (formerly YlaN) was demonstrated to be essential in Bacillus subtilis unless excess Fe is added to the growth medium, suggesting a role in Fe homeostasis. Here, we demonstrate that Fpa is essential in S. aureus upon Fe deprivation. Null fur alleles bypassed the essentiality of Fpa. The absence of Fpa abolished the derepression of Fur-regulated genes during Fe limitation. Bioinformatic analyses suggest that fpa was recruited to Gram-positive bacteria and, once acquired, was maintained in the genome as it co-evolved with Fur. Consistent with a role for Fpa in alleviating Fur-dependent repression, Fpa and Fur interacted in vivo, and Fpa decreased the DNA-binding ability of Fur in vitro. Fpa bound Fe(II) in vitro using oxygen or nitrogen ligands with an association constant that is consistent with a physiological role in Fe homeostasis. These findings have led to a model wherein Fpa is an Fe(II) binding protein that influences Fur-dependent regulation through direct interaction.IMPORTANCEIron (Fe) is an essential nutrient for nearly all organisms. If Fe homeostasis is not maintained, Fe may accumulate in the cytosol, which can be toxic. Questions remain about how cells efficiently balance Fe uptake and usage to prevent overload. Iron uptake and proper metalation of proteins are essential processes in the mammalian bacterial pathogen Staphylococcus aureus. Understanding the gene products involved in the genetic regulation of Fe uptake and usage and the physiological adaptations that S. aureus uses to survive in Fe-depleted conditions provides insight into pathogenesis. Herein, we demonstrate that the DNA-binding activity of the ferric uptake regulator transcriptional repressor is alleviated under Fe limitation, but uniquely, in S. aureus, alleviation requires the presence of Fpa.

Nitrification is a core process in the global nitrogen (N) cycle mediated by ammonia-oxidizing microorganisms, including ammonia-oxidizing archaea (AOA) as a key player. Although much is known about AOA abundance and diversity across environments, the genetic drivers of the ecophysiological adaptations of the AOA are often less clearly defined. This is especially true for AOA within the genus Nitrosocosmicus, which have several unique physiological traits (e.g., high substrate tolerance, low substrate affinity, and large cell size). To better understand what separates the physiology of Nitrosocosmicus AOA, we performed comparative genomics with genomes from 39 cultured AOA, including five Nitrosocosmicus AOA. The absence of a canonical high-affinity type ammonium transporter and typical S-layer structural genes was found to be conserved across all Nitrosocosmicus AOA. In agreement, cryo-electron tomography confirmed the absence of a visible outermost S-layer structure, which has been observed in other AOA. In contrast to other AOA, the cryo-electron tomography highlighted the possibility that Nitrosocosmicus AOA may possess a glycoprotein or glycolipid-based glycocalyx cell covering outer layer. Together, the genomic, physiological, and metabolic properties revealed in this study provide insight into niche adaptation mechanisms and the overall ecophysiology of members of the Nitrosocosmicus clade in various terrestrial ecosystems.

Importance: Nitrification is a vital process within the global biogeochemical nitrogen cycle but plays a significant role in the eutrophication of aquatic ecosystems and the production of the greenhouse gas nitrous oxide (N₂O) from industrial agriculture ecosystems. While various types of ammonia-oxidizing microorganisms play a critical role in the N cycle, ammonia-oxidizing archaea (AOA) are often the most abundant nitrifiers in natural environments. Members of the genus Nitrosocosmicus are one of the prevalent AOA groups detected in undisturbed terrestrial ecosystems and have previously been reported to possess a range of physiological characteristics that set their physiology apart from other AOA species. This study provides significant progress in understanding these unique physiological traits and their genetic drivers. Our results highlight how physiological studies based on comparative genomics-driven hypotheses can contribute to understanding the unique niche of Nitrosocosmicus AOA.

Gene expression and proper downstream cellular functions upon facing environmental shifts depend on the combined and cooperative regulation of genetic networks. Here, we identified cAMP receptor protein (CRP) as a master regulator of (p)ppGpp (guanosine tetra- and penta-phosphate) homeostasis. Via CRP-mediated direct transcriptional regulation of the (p)ppGpp synthetase/hydrolase RelA and SpoT, cAMP-CRP stimulates pervasive accumulation of (p)ppGpp under glucose-limiting conditions. Notably, CRP exerts a nonclassical property as a translational regulator through YfiQ-dependent acetylation of ribosome protein S1 at K247, which further enhances the translation of RelA, SpoT, and CRP itself. From a synthetic biology perspective, this self-activating feedback loop for (p)ppGpp synthesis highlights the function of CRP-mediated dual enhancement (CMDE) in controlling bacterial gene expression, which enables stable activation of genetic circuits. CMDE applied in synthetic circuits leads to a stable increase in p-coumaric acid, cinnamic acid, and pinosylvin production. Our findings showed that CRP-mediated dual circuits for (p)ppGpp regulation enable robust activation that could address bioproduction and other biotechnological needs.IMPORTANCETranscriptional-translational coordination is fundamental for rapid and efficient gene expression in most bacteria. Here, we uncovered the roles of cAMP-CRP in this process. We found that CRP distinctly increases RelA and SpoT transcription and translation, and that acetylation of S1 at K247 accelerates the self-activation of the leading CRP under glucose-limiting conditions. We further found that elevated (p)ppGpp significantly impedes the formation of the cAMP-CRP complex, an active form responsible for transcriptional activation. A model was created in which cAMP-CRP and (p)ppGpp cooperate to dynamically modulate the efficiency of transcriptional-translational coordination responses to stress. More broadly, productive activation in synthetic circuits was achieved through the application of CRP-mediated dual enhancement (CMDE), promising to inspire new approaches for the development of cell-based biotechnologies.

Parasites from the Leishmania genus are the causative agents of leishmaniasis and primarily reside within macrophages during mammalian infection. Their ability to establish intracellular infection provides a secure niche for proliferation while evading detection. However, successful multiplication within mammalian cells requires the orchestration of multiple mechanisms that control host cell viability. In contrast, innate immune cells, such as macrophages, can undergo different forms of cell death in response to pathogenic intracellular microbes. Thus, modulation of these different forms of host cell death is crucial for Leishmaniasis development. The regulation of host cell apoptosis, a form of programmed cell death, is crucial for sustaining parasites within viable host cells. Accordingly, several studies have demonstrated evasion of apoptosis induced by dermotropic and viscerotropic Leishmania species. Conversely, the prevention of pyroptosis, an inflammatory form of cell death, ensures the establishment of infection by silencing the release of mediators that could trigger massive proinflammatory responses. This manuscript explores how Leishmania regulates various host cell death pathways and overviews seminal studies on regulating host cell apoptosis by different Leishmania species.

Microbial processes operate at the microscale, which is not resolved by existing ecosystem models. Here, we present a novel model that simulates a 1 mL three-dimensional cube using a hybrid Lagrangian-Eulerian approach, at ecologically relevant timescales. The model simulates individual microbes, including three phytoplankton size classes with healthy, senescent, and dead lifecycle stages; copiotrophic and oligotrophic heterotrophic bacteria; and dissolved organic matter at 50 µm resolution. Diffusion, shear, sedimentation, chemotaxis, and attachment processes are explicitly resolved. The emerging quantitative representation of the ecosystem shows that (1) copiotrophs grow mostly attached to eukaryotic phytoplankters and get almost all of their carbon from them vs. oligotrophs that grow on exudates and lysates of cyanobacteria; (2) contrasting diel patterns in substrate appearance in the phycosphere vs. ambient water and growth of particle-associated copiotrophs vs. free-living oligotrophs; (3) attached bacteria reduce carbon flux from the phycosphere, lowering chemotactic efficiency toward eukaryotes below that toward cyanobacteria; (4) shear reduces chemotactic efficiency and fitness of the copiotroph; and (5) the main benefit of chemotaxis is to locate attachment partners. These patterns are consistent with available observations. Our study provides insights into the microscale ecology of marine bacteria, and the open-source code is a tool for further research in this area.IMPORTANCEA large amount of global CO₂ fixation is performed by marine phytoplankton, and a substantial fraction of that is released as dissolved organic carbon and further processed by heterotrophic bacteria. The interaction between phytoplankton and bacteria, i.e., the carbon flux between them, is therefore an important process in the global carbon and climate system. Some bacteria have developed specialized behavioral traits, like swimming and attachment, to increase their carbon acquisition. These interactions occur at the micrometer scale, for example, the immediate vicinity of phytoplankters (the phycosphere), but existing biogeochemical models typically only simulate down to the 1 meter vertical or ~100 kilometer horizontal scale. We present a new microscale model and use it to predict fluxes and other features in the surface ocean. The model makes important predictions about the fluxes between various types of phytoplankton and bacteria and the role of behavioral traits, and it provides a basis and tool for further research in this area.

The current treatments for toxoplasmosis are only active against fast-growing tachyzoites, present in acute infections, with little effect on slow-growing bradyzoites within tissue cysts, present in latent chronic infections. The mitochondrion of Toxoplasma gondii is essential for its survival, and one of the major anti-parasitic drugs, atovaquone, inhibits the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase site. Coenzyme Q (also known as ubiquinone [UQ]) consists of a quinone head and a lipophilic, isoprenoid tail that anchors UQ to membranes. The synthesis of the isoprenoid unit is essential for cell growth and is inhibited by lipophilic bisphosphonates, which inhibit the parasite growth. In this work, we investigated the effect of lipophilic bisphosphonates on the chronic stages of T. gondii. We discovered that three lipophilic bisphosphonates (BPH-1218, BPH-1236, and BPH-1238), effective for the acute infection, were also effective in controlling the development of chronic stages. We showed effectiveness by testing them against in vitro cysts and in vivo derived tissue cysts and, most importantly, these compounds reduced the cyst burden in the brains of chronically infected mice. We monitored the activity of infected mice non-invasively and continuously with a novel device termed the CageDot. A decrease in activity accompanied the acute phase, but mice recovered to normal activity and showed signs of hyperactivity when the chronic infection was established. Moreover, treatment with atovaquone or BPH-1218 ameliorated the hyperactivity observed during the chronic infection.IMPORTANCETreatment for toxoplasmosis is challenged by a lack of effective drugs to eradicate the chronic stages. Most of the drugs currently used are poorly distributed to the central nervous system, and they trigger allergic reactions in a large number of patients. There is a compelling need for safe and effective treatments for toxoplasmosis. Bisphosphonates (BPs) are analogs of inorganic pyrophosphate and are used for the treatment of bone disorders. BPs target the isoprenoid pathway and are effective against several experimental parasitic infections. Some lipophilic BPs can specifically inhibit the mitochondrial activity of Toxoplasma gondii by interfering with the mechanism by which ubiquinone is inserted into the inner mitochondrial membrane. In this work, we present the effect of three lipophilic BPs against T. gondii chronic stages. We also present a new strategy for the monitoring of animal activity during disease and treatment that is non-invasive and continuous.

Gammaherpesviruses are species-specific, ubiquitous pathogens that establish lifelong infection in their hosts and are associated with cancers, including B cell lymphomas. Type I and II interferons (IFNs) are critical for the control of acute and chronic gammaherpesvirus infection. However, the cell type-specific role of IFN signaling during natural infection is poorly defined and is masked by the altered viral pathogenesis observed in hosts with global IFN deficiencies. STAT1 is a constitutively expressed transcription factor that is critical for the effector function of type I and II IFNs. In this study, we defined the impact of B cell-specific STAT1 expression on gammaherpesvirus infection using murine gammaherpesvirus 68 (MHV68). While the acute stage of MHV68 infection was not affected, we found opposite, anatomic site-dependent effects of B cell-intrinsic STAT1 expression during chronic infection. Consistent with the antiviral role of STAT1, B cell-specific STAT1 expression attenuated the latent viral reservoir in peritoneal B cells of chronically infected mice. In contrast, STAT1 expression in splenic B cells supported the establishment of the latent MHV68 reservoir in germinal center B cells, revealing an unexpected proviral role of B cell-intrinsic STAT1 expression during chronic gammaherpesvirus infection. These STAT1-dependent MHV68 chronic infection phenotypes were fully recapitulated in the peritoneal cavity but not the spleen of mice with B cell-specific deficiency of type I IFN receptor. In summary, our study uncovers the intriguing combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression during chronic gammaherpesvirus infection.IMPORTANCEInterferons (IFNs) execute broadly antiviral roles during acute and chronic viral infections. The constitutively expressed transcription factor STAT1 is a critical downstream effector of IFN signaling. Our studies demonstrate that B cell-intrinsic STAT1 expression has opposing and anatomic site-dependent roles during chronic gammaherpesvirus infection. Specifically, B cell-intrinsic STAT1 expression restricted gammaherpesvirus latent reservoir in the peritoneal cavity, consistent with the classical antiviral role of STAT1. In contrast, decreased STAT1 expression in splenic B cells led to the attenuated establishment of gammaherpesvirus latency and decreased latent infection of germinal center B cells, highlighting a novel proviral role of B cell-intrinsic STAT1 expression during chronic infection with a B cell-tropic gammaherpesvirus. Interestingly, B cell-specific type I IFN receptor deficiency primarily recapitulated the antiviral role of B cell-intrinsic STAT1 expression, suggesting the compensatory function of B cell-intrinsic type II IFN signaling or an IFN-independent proviral role of B cell-intrinsic STAT1 expression during chronic gammaherpesvirus infection.