mBio最新文献

Na⁺-K⁺-ATPase alpha subunit 1 (ATP1A1) is the main functional part of the sodium pump. In addition to the well-known role in ion transport, it also acts as a signal transducer. Porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) utilizes multiple entry factors and pathways to initiate infection, posing a significant challenge to the global swine industry. However, the molecules conferring PRRSV-2 infection have not been fully characterized. Here, ATP1A1 is identified as a novel factor in PRRSV-2 attachment and internalization. ATP1A1 formed clusters in the plasma membrane very early following PRRSV-2 infection and co-internalizes with virions. Knockdown of ATP1A1 significantly suppressed PRRSV-2 infection by reducing viral attachment, and the specific chemical ligands, ouabain and PST2238, effectively reduced viral internalization without affecting viral attachment, leading to decreased viral infection. Mechanically, ATP1A1-Src signaling-dependent activation of EGFR and caveolin-1 was required for efficient PRRSV-2 uptake through macropinocytosis and caveolae/raft-mediated endocytosis. Furthermore, internalized virions were subsequently trafficked to ATP1A1/CD163-positive early endosomes, where uncoating occurs. In detail, PRRSV glycoprotein 4 (GP4), a major determinant for viral cellular tropism, was found to interact with the fourth extracellular region (ER4) of ATP1A1, dependent on its C-terminus. A synthetic ATP1A1-ER4 peptide inhibited PRRSV-2 replication by competitively reducing viral attachment and internalization in a dose-dependent manner. Most importantly, a specific nanobody targeting ATP1A1-ER4 provided broad inhibition against various PRRSV-2 lineages both in PAMs and Marc-145 cells. Collectively, these results elucidate that ATP1A1 is important for PRRSV-2 attachment and internalization, offering a potential target for the development of antiviral treatments.

Importance: PRRSV continues to cause severe financial losses to the global swine industry. It is feasible to develop safe and effective antiviral strategies based on the initial step of viral infection, that is, the recognition of the virus by the cellular entry factors. However, the interactions between PRRSV and host factors initiating viral attachment and internalization are not fully understood yet. In this study, ATP1A1 was identified to promote both PRRSV-2 attachment and internalization through macropinocytosis and caveolae/raft-dependent endocytosis. These findings reveal an unrecognized entry mechanism of PRRSV-2 and provide novel insights for the development of antiviral drugs and vaccines against the virus.

Despite effective suppression of viremia by antiretroviral therapy, HIV-1 persists in long-lived cellular reservoirs. Novel approaches aimed at eliminating these reservoirs are therefore essential for an HIV-1 cure. Among emerging cure strategies, harnessing antibody-dependent cellular cytotoxicity (ADCC) has generated significant interest. In this mGem, we discuss how small CD4-mimetic compounds (CD4mc), by forcing envelope (Env) into more "open" conformations, thereby exposing conserved CD4-induced epitopes, can unlock the ADCC potential of non-neutralizing antibodies. We also highlight how type I interferons complement this approach by upregulating BST-2, thereby increasing Env at the cell surface, diminishing Vpu-mediated immune evasion, and enhancing NK cell effector functions. Together, these synergistic interventions provide a promising framework to improve immune recognition of infected cells and potentially reduce the size of the HIV-1 reservoir.

Coronavirus infections can trigger multiple modes of cell death, leading to severe infectious diseases. The process is modulated by host factors with mechanisms yet to be fully elucidated. Here, we first demonstrated that the host factor A20 regulated PANoptosis during porcine deltacoronavirus (PDCoV) infection, thereby contributing to the antiviral defense response. We found that PDCoV could induce PANoptosis in intestinal epithelial cells, which facilitates the extracellular release of viral particles through this form of programmed cell death. A20 restricted the PANoptosome assembly and downstream death signaling by targeting RIPK3 ubiquitin chains for degradation. Consequently, loss of A20 exacerbated cell lysis and enhanced the release of viral particles, although this effect does not alter viral entry or replication. We further established that PDCoV-induced PANoptosis-dependent release was driven by osmotic imbalance resulting from membrane pore formation mediated by GSDMD and MLKL, rather than by direct transmembrane egress of viral particles. Transwell models showed that pharmacological inhibition of the pore-forming activities of GSDMD and MLKL reduced viral dissemination and preserved epithelial barrier integrity. These findings advance our understanding of enteric coronavirus pathogenesis and suggest that the A20-PANoptosis axis represents a potential target for antiviral intervention.IMPORTANCECoronaviruses have repeatedly posed significant threats to both human and animal health. Here, we used porcine deltacoronavirus (PDCoV), a highly enterotropic zoonotic pathogen, to uncover a novel mechanism by which coronaviruses exploit PANoptosis to facilitate viral egress. We demonstrate that PDCoV infection triggers PANoptosis in intestinal epithelial cells, leading to plasma membrane rupture and subsequent viral release. Importantly, we identified the host ubiquitin-editing enzyme A20 as a critical negative regulator of this process. A20 restricts PANoptosome assembly by specifically deubiquitinating RIPK3, thereby limiting cell lysis and suppressing viral dissemination without affecting viral replication. Our findings offer fundamental insights into coronavirus-host interactions and highlight the therapeutic potential of targeting lytic cell death to combat viral dissemination.

Multicellular life arose in a world dominated by microorganisms, a reality that has imposed a constant and pervasive selective pressure on all subsequent complex organisms. The immune system has been historically defined by its role in pathogen clearance through resistance mechanisms. However, a complementary and equally critical strategy is to enable the peaceful and inevitable coexistence with microorganisms, allowing each host species to shelter a unique associated microbiome. The term tolerance holds multiple meanings in immunology, yet all underlie a balanced and cooperative host-microorganism relationship. Each represents a different aspect of how the immune system limits tissue damage while maintaining functionality in the presence of microbial or inflammatory stimuli. Using the intestinal mucosa as a paradigm, we explore how epithelial barrier integrity, toxin neutralization, tissue repair, and stress response underpin disease tolerance; how microbial exposure calibrates innate immunity via epigenetic and metabolic reprogramming (LPS tolerance); and how the gut microenvironment fosters the generation of tolerogenic antigen-presenting cells and microbe-specific regulatory T cells to enforce immunological tolerance. We further explore how the microbiota itself is a potent inducer of these tolerogenic pathways and highlight IL-10 as a major hub, connecting different tolerogenic circuits. Finally, we examine the hygiene hypothesis, arguing that lifestyle changes during the Anthropocene disrupt these finely tuned tolerance mechanisms, thereby contributing to the rising incidence of immune-mediated diseases. We posit that these tolerance programs are fundamental prerequisites for engendering host-microbiota symbiosis, a relationship forged over millennia of co-evolution and endangered in the contemporary world.

Salmonella enterica utilizes a virulence-associated type III secretion system (T3SS) to inject bacterial effectors directly into host cells. Central to this machinery is the sorting platform (SP), a cytosolic assembly whose core scaffolding protein, SpaO, is produced in two isoforms: a full-length (SpaO^L) and a shorter variant (SpaO^short) comprising the C-terminal 101 residues of SpaO^L. Although SpaO^short is evolutionarily conserved across type III secretion systems, its precise function has remained elusive. Here, we combined a sensitive, real-time translocation assay with site-directed photo-crosslinking to inform the role of SpaO^short in Salmonella SPI-1 T3SS. Quantitative translocation data show that while SpaO^short is not absolutely required for effector translocation, its absence significantly dampens T3SS-mediated protein delivery. Biochemical and structural probing further defined the interfaces between SpaO^L and SpaO^short, uncovering a previously unrecognized interaction mode between the two isoforms. Photo-crosslinking revealed that a single SpaO^L molecule accommodates a SpaO^short dimer via an N-terminal "docking motif," an interaction that occurs in vivo while SpaO^L is associated with other SP components. These results support a model in which SpaO^short is integrated into the SP pods alongside SpaO^L, OrgA, and OrgB, likely contributing to pod stabilization. Collectively, these findings provide new insights into how Salmonella and related bacteria assemble and maintain these specialized protein-injection systems.IMPORTANCESalmonella enterica is an increasing global public health threat. As part of its virulence arsenal, Salmonella relies on a type III secretion system (T3SS) or injectisome, a molecular injection device that translocates effector proteins into host cells to promote invasion and inflammation. A central component of this machine is the SpaO protein, which is produced in two forms: a full-length form and a shorter variant. Here, by studying the functional and structural relationship between the two SpaO forms in their native cellular environment, we define how and when they assemble within the injectisome. Employing quantitative injection assays in cultured cells, we define the shorter SpaO variant as an accessory structural piece that boosts effector delivery. These findings refine our understanding of injectisome assembly and function and provide mechanistic insight to inform future efforts to target T3SS-dependent pathogens through antivirulence strategies.

In laboratory settings, bacteria grow in static culture with more nutrients than they require. However, bacteria in nature experience flowing environments that are nutrient-limited. Using microfluidics and single-cell imaging, we discover that shear flow promotes growth of the human pathogens Pseudomonas aeruginosa and Vibrio cholerae at surprisingly low nutrient concentrations. In static environments, cells require high nutrient concentrations as they steadily consume non-renewable resources. In slower-flowing environments, cells grow and deplete nutrients, which generates spatial gradient profiles. In faster-flowing environments, cells grow robustly and form microcolonies even at very low concentrations due to rapid nutrient replenishment. By precisely delivering nutrients using microfluidics, we learned that cells in flow can grow on glucose concentrations 1,000 times lower than those observed in typical laboratory experiments. The ultralow glucose concentrations sufficient for growth in flow closely align with the affinity of bacterial glucose transporters, suggesting that bacteria have evolved in flowing environments with scarce nutrients. Collectively, our results emphasize the limits of traditional culturing approaches and highlight how shear flow can promote bacterial growth and shape spatial gradients.IMPORTANCEWhile bacteria in nature experience flow, laboratory conditions typically omit flow. Additionally, bacteria in nature are often nutrient-limited, but laboratory conditions contain excess nutrients. Here, we use microfluidic technology to determine how flow impacts growth of a bacterial pathogen under nutrient limitation. We discover that flow sustains growth at glucose concentrations 1,000 times lower than traditionally observed. In traditional experiments, bacteria grow on a high concentration of a non-renewable resource. In our microfluidic experiments, bacteria can grow on surprisingly low concentrations of resources if they are renewed by flow. Our results emphasize the need to study bacteria in realistic contexts and suggest that scientists should rethink how cells experience nutrient limitation in nature.

Schizosaccharomyces pombe adapts to phosphate starvation by upregulating the expression of (i) a cell-surface acid phosphatase, Pho1, that mobilizes inorganic phosphate from the extracellular milieu; (ii) transmembrane transporters that take up inorganic phosphate (Pho84, Pho841, and Pho842) and glycerophosphocholine (Tgp1); and (iii) secreted extracellular 5'-nucleotidase enzymes (Efn1 and Efn2) that release inorganic phosphate from rNMPs, with a preference for CMP. The expression of SPAC24B11.05, a fission yeast homolog of the budding yeast 5'-nucleotidase Sdt1, is upregulated during phosphate starvation, and the protein accumulates without being secreted. Here, we characterized recombinant SPAC24B11.05 (herein Ifn1, for intracellular 5'-nucleotidase) as a Mg²⁺-dependent phosphohydrolase of the aspartyl-phosphatase (HAD) superfamily. Unlike Sdt1, which is specific for pyrimidine mononucleotides and nicotinamide mononucleotide (NMN), Ifn1 displays a preference for hydrolysis of GMP > IMP > CMP > AMP > UMP and is unable to hydrolyze NMN. Ifn1 activity is abolished by alanine mutations of the Asp11 nucleophile of the signature ¹¹DLDNC¹⁵ motif and by alanines in lieu of Asp80 and Asp174 that are predicted to coordinate the ribose hydroxyls and the metal cofactor, respectively. Changing Ifn1 Arg50, which is predicted to engage the guanine nucleobase, to Asn, the corresponding residue in Sdt1, enhances hydrolysis of CMP and AMP and suppresses hydrolysis of GMP, IMP, and UMP, with no gain of activity with NMN. We find that overexpression of catalytically active Ifn1 is toxic to fission yeast.IMPORTANCEPhosphate starvation in fission yeast triggers increased expression of enzymes with imputed roles in phosphate dynamics. Many starvation-induced phosphohydrolases are annotated as acting on nucleotides, though their substrate specificities have not been interrogated. Here, we characterize fission yeast Ifn1 as a starvation-induced 5'-nucleotidase of the aspartyl-phosphatase (HAD) superfamily with a preference for hydrolysis of GMP and IMP that distinguishes it from the homologous budding yeast pyrimidine-specific 5'-nucleotidase Sdt1. A single swap of Ifn1 Arg50 to Asn (the equivalent position in Sdt1) elicits a substrate switch, manifested as a gain of activity with CMP and suppression of activity with GMP and IMP. An emergent theme is that 5'-nucleotidase substrate specificity is a tunable property.

Reproductive-aged females mount stronger antibody responses to influenza vaccination than males, with the sex difference waning in older age. Estradiol has been implicated as a driver, but the mechanisms mediating how estradiol affects B cell function remain elusive. Adult (3 months) and aged (17 months) male and female mice were vaccinated and boosted with inactivated influenza vaccine. Metabolomics analysis of splenic B cells revealed that adult female B cells were enriched in lipid metabolic pathways, whereas B cells from males were enriched in central carbon-associated pathways following vaccination. B cells from vaccinated adult females exhibited greater expression of mTOR and related proteins than those from males, a difference diminished in aged mice. In adult females, estradiol depletion reduced, and replacement increased, mTOR activity in B cells, particularly in germinal center B cells and plasmablasts in lymphoid tissues, and plasma cells in bone marrow. In males, neither testosterone depletion nor repletion altered B cell metabolism. These findings are consistent with evidence that estradiol enhances mTOR activation via estrogen receptor α (ERα) signaling, suggesting coordinated regulation between estrogen and mTOR signaling in B cells. Inhibition of mTOR with rapamycin impaired vaccine-induced antibody responses and protection in adult females. In aged females, supplementation with estradiol or treatment with a selective ERα agonist increased mTOR signaling and enhanced antibody responses compared with mock-treated aged females. These data identify estrogen signaling as a regulator of B cell metabolism that supports greater expansion and function of antibody-secreting cells following vaccination in females compared with males.

Importance: Vaccine-induced immunity differs between the sexes, with adult females mounting stronger antibody responses to influenza vaccination than age-matched males. We show that estradiol in females regulates B cell metabolism to promote the maturation and metabolic activation of antibody-secreting B cells, thereby enhancing humoral immunity and protection following vaccination. mTOR signaling in B cells was greater in adult females than males after vaccination, which was diminished with aging or depletion of estradiol. Therapeutic treatment of aged females with either estradiol or a selective estrogen receptor α modulator increased mTOR signaling and improved vaccine-induced antibody responses, thereby eliminating the effects of aging on influenza immunity. Harnessing estrogen-signaling mechanisms to improve responses to influenza vaccines could be a novel therapeutic strategy to improve public health.

An outbreak of H3N1 low-pathogenic avian influenza virus (LPAIV) in Belgium in 2019 caused unexpected levels of mortality and morbidity in poultry. These viruses possess an NA polymorphism associated with plasminogen (PLG) binding, as well as an atypical sequence around the HA cleavage site; accordingly, HA cleavage mediated by NA-driven PLG recruitment has been proposed to underlie their systemic spread and pathogenicity. To test this, we established a reverse genetics system for A/chicken/Belgium/460/2019 and created single mutations in HA (K345R) and NA (S122N) that restored the viruses to normal consensus, as well as an HA/NA double mutant. Confirming previous work, trypsin-independent spread and HA cleavage of wild-type Ck/Belgium were observed in the presence of fetal bovine serum containing PLG in vitro. Dose-dependent HA cleavage and trypsin-independent spread were also observed in the presence of purified chicken PLG. Compared to the wild-type virus, both HA cleavage and virus spread in vitro were reduced by the HA K345R mutation and further blocked by the NA mutation S122N. PLG-mediated HA cleavage was seen in a variety of avian cell lines and chicken organoids, excluding cell type-dependent effects. Furthermore, in ovo tests showed that mutant viruses unable to recruit PLG were less able to replicate systemically in chicken embryos. Bioinformatics analyses revealed other viruses that could potentially recruit PLG, including two independent outbreaks of H6N1 viruses, one of which we confirmed PLG-driven spread in vitro. We conclude that PLG recruitment by NA is a general virulence mechanism of N1 LPAIVs.IMPORTANCEAvian influenza viruses are divided into high or low pathogenicity based on the sequence of their hemagglutinin (HA) and their lethality in chickens. The majority of AIV strains circulating in the wild are of low pathogenicity both in waterfowl and when they spill over into domestic poultry. However, some low-pathogenicity strains can cause serious disease in poultry. A severe 2019 outbreak of an H3N1 strain has been suggested to result from the viral neuraminidase (NA) recruiting cellular plasminogen to proteolytically activate HA. Here, we confirmed that the sequence of the NA at position 122 is the primary determinant of plasminogen-driven HA cleavage, but that the unusual sequence at the HA cleavage also contributes to pathogenicity. Furthermore, we show that this N1 NA sequence motif can be used to identify other unexpectedly virulent AIV strains. This work, therefore, adds to our ability to risk assess AIV strains from sequence-based surveillance.