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The interaction of neutrophils with T cells has been the subject of debate and controversies. Previous studies have suggested that neutrophils may suppress or activate T cells. Despite these studies, the interaction between neutrophils and T cells has remained a largely unexplored field. Here, based on our RNA sequencing (RNA-seq) analysis, we found that neutrophils have differential transcriptional and functional profiling depending on the CD4 T-cell count of the HIV-infected individual. In particular, we identified that neutrophils in healthy individuals express surface Galectin-9 (Gal-9), which is down-regulated upon activation, and is consistently down-regulated in HIV-infected individuals. However, down-regulation of Gal-9 was associated with CD4 T-cell count of patients. Unstimulated neutrophils express high levels of surface Gal-9 that is bound to CD44, and, upon stimulation, neutrophils depalmitoylate CD44 and induce its movement out of the lipid raft. This process causes the release of Gal-9 from the surface of neutrophils. In addition, we found that neutrophil-derived exogenous Gal-9 binds to cell surface CD44 on T cells, which promotes LCK activation and subsequently enhances T-cell activation. Furthermore, this process was regulated by glycolysis and can be inhibited by interleukin (IL)-10. Together, our data reveal a novel mechanism of Gal-9 shedding from the surface of neutrophils. This could explain elevated plasma Gal-9 levels in HIV-infected individuals as an underlying mechanism of the well-characterized chronic immune activation in HIV infection. This study provides a novel role for the Gal-9 shedding from neutrophils. We anticipate that our results will spark renewed investigation into the role of neutrophils in T-cell activation in other acute and chronic conditions, as well as improved strategies for modulating Gal-9 shedding., This study shows that HIV-infected individuals have different neutrophil profiles depending on their CD4 T cell count. In particular, neutrophils express high levels of surface Gal-9 but this is shed upon stimulation; this exogenous Gal-9 binds to CD44 on T cells, which promotes LCK activation and subsequently enhances T cell activation.

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing., Little is known about how membrane fission occurs in bacteria; this study suggests that the membrane fission protein FisB exploits the unique cellular geometry encountered during sporulation to enable its localization to the fission site through a novel mechanism, where it catalyzes membrane scission.

The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.

Microbial conversion of dietary or drug substrates into small bioactive molecules represents a regulatory mechanism by which the gut microbiota alters intestinal physiology. Here, we show that a wide variety of gut bacteria can metabolize the dietary supplement and antidepressant 5-hydroxytryptophan (5-HTP) to 5-hydroxyindole (5-HI) via the tryptophanase (TnaA) enzyme. Oral administration of 5-HTP results in detection of 5-HI in fecal samples of healthy volunteers with interindividual variation. The production of 5-HI is inhibited upon pH reduction in in vitro studies. When administered orally in rats, 5-HI significantly accelerates the total gut transit time (TGTT). Deciphering the underlying mechanisms of action reveals that 5-HI accelerates gut contractility via activation of L-type calcium channels located on the colonic smooth muscle cells. Moreover, 5-HI stimulation of a cell line model of intestinal enterochromaffin cells results in significant increase in serotonin production. Together, our findings support a role for bacterial metabolism in altering gut motility and lay the foundation for microbiota-targeted interventions.

Zoonotic avian influenza viruses pose severe health threats to humans. Of several viral subtypes reported, the low pathogenic avian influenza H7N9 virus has since February 2013 caused more than 1,500 cases of human infection with an almost 40% case-fatality rate. Vaccination of poultry appears to reduce human infections. However, the emergence of highly pathogenic strains has increased concerns about H7N9 pandemics. To develop an efficacious H7N9 human vaccine, we designed vaccine viruses by changing the patterns of N-linked glycosylation (NLG) on the viral hemagglutinin (HA) protein based on evolutionary patterns of H7 HA NLG changes. Notably, a virus in which 2 NLG modifications were added to HA showed higher growth rates in cell culture and elicited more cross-reactive antibodies than did other vaccine viruses with no change in the viral antigenicity. Developed into an inactivated vaccine formulation, the vaccine virus with 2 HA NLG additions exhibited much better protective efficacy against lethal viral challenge in mice than did a vaccine candidate with wild-type (WT) HA by reducing viral replication in the lungs. In a ferret model, the 2 NLG-added vaccine viruses also induced hemagglutination-inhibiting antibodies and significantly suppressed viral replication in the upper and lower respiratory tracts compared with the WT HA vaccines. In a mode of action study, the HA NLG modification appeared to increase HA protein contents incorporated into viral particles, which would be successfully translated to improve vaccine efficacy. These results suggest the strong potential of HA NLG modifications in designing avian influenza vaccines., This study shows that changing the pattern of N-glycosylation of the pathogenic avian influenza H7N9 virus hemagglutinin protein increases the amount of hemagglutinin incorporated into the viral membrane; the candidate vaccine virus induces neutralizing antibodies and protects animal models from lethal viral challenge.

Although dysregulation of mitochondrial dynamics has been linked to cellular senescence, which contributes to advanced age-related disorders, it is unclear how Krüppel-like factor 5 (Klf5), an essential transcriptional factor of cardiovascular remodeling, mediates the link between mitochondrial dynamics and vascular smooth muscle cell (VSMC) senescence. Here, we show that Klf5 down-regulation in VSMCs is correlated with rupture of abdominal aortic aneurysm (AAA), an age-related vascular disease. Mice lacking Klf5 in VSMCs exacerbate vascular senescence and progression of angiotensin II (Ang II)–induced AAA by facilitating reactive oxygen species (ROS) formation. Klf5 knockdown enhances, while Klf5 overexpression suppresses mitochondrial fission. Mechanistically, Klf5 activates eukaryotic translation initiation factor 5a (eIF5a) transcription through binding to the promoter of eIF5a, which in turn preserves mitochondrial integrity by interacting with mitofusin 1 (Mfn1). Accordingly, decreased expression of eIF5a elicited by Klf5 down-regulation leads to mitochondrial fission and excessive ROS production. Inhibition of mitochondrial fission decreases ROS production and VSMC senescence. Our studies provide a potential therapeutic target for age-related vascular disorders., Down-regulation of Klf5, an essential transcriptional factor of cardiovascular remodeling, in vascular smooth muscle cells is correlated with vascular senescence and rupture of abdominal aortic aneurysm. This effect is mediated by increased mitochondrial fission and production of reactive oxygen species.

Rapid antibiotic susceptibility testing (AST) for Neisseria gonorrhoeae (Ng) is critically needed to counter widespread antibiotic resistance. Detection of nucleic acids in genotypic AST can be rapid, but it has not been successful for β-lactams (the largest antibiotic class used to treat Ng). Rapid phenotypic AST for Ng is challenged by the pathogen’s slow doubling time and the lack of methods to quickly quantify the pathogen’s response to β-lactams. Here, we asked two questions: (1) Is it possible to use nucleic acid quantification to measure the β-lactam susceptibility phenotype of Ng very rapidly, using antibiotic-exposure times much shorter than the 1- to 2-h doubling time of Ng? (2) Would such short-term antibiotic exposures predict the antibiotic resistance profile of Ng measured by plate growth assays over multiple days? To answer these questions, we devised an innovative approach for performing a rapid phenotypic AST that measures DNA accessibility to exogenous nucleases after exposure to β-lactams (termed nuclease-accessibility AST [nuc-aAST]). We showed that DNA in antibiotic-susceptible cells has increased accessibility upon exposure to β-lactams and that a judiciously chosen surfactant permeabilized the outer membrane and enhanced this effect. We tested penicillin, cefixime, and ceftriaxone and found good agreement between the results of the nuc-aAST after 15–30 min of antibiotic exposure and the results of the gold-standard culture-based AST measured over days. These results provide a new pathway toward developing a critically needed phenotypic AST for Ng and additional global-health threats., This study presents a new approach for testing antibiotic-susceptibility in the slow-growing human pathogen Neisseria gonorrhoeae, based on measuring accessibility of pathogen nucleic acids to extracellular reagents. Surprisingly, the beta-lactam susceptibility phenotype was revealed with antibiotic exposures shorter than cell division.

The gastric pathogen Helicobacter pylori requires a noncanonical cytosolic chemoreceptor transducer-like protein D (TlpD) for efficient colonization of the mammalian stomach. Here, we reconstituted a complete chemotransduction signaling complex in vitro with TlpD and the chemotaxis (Che) proteins CheW and CheA, enabling quantitative assays for potential chemotaxis ligands. We found that TlpD is selectively sensitive at micromolar concentrations to bleach (hypochlorous acid, HOCl), a potent antimicrobial produced by neutrophil myeloperoxidase during inflammation. HOCl acts as a chemoattractant by reversibly oxidizing a conserved cysteine within a 3His/1Cys Zn-binding motif in TlpD that inactivates the chemotransduction signaling complex. We found that H. pylori is resistant to killing by millimolar concentrations of HOCl and responds to HOCl in the micromolar range by increasing its smooth-swimming behavior, leading to chemoattraction to HOCl sources. We show related protein domains from Salmonella enterica and Escherichia coli possess similar reactivity toward HOCl. We propose that this family of proteins enables host-associated bacteria to sense sites of tissue inflammation, a strategy that H. pylori uses to aid in colonizing and persisting in inflamed gastric tissue., A pathogenic bacteria is found to be attracted to bleach. Helicobacter pylori, notorious for causing stomach ulcers and cancer, senses bleach as an attractant, enabling it to localize to sites of inflamed gastric tissue (hypochlorous acid, the active agent of bleach, is produced by host neutrophils as an antimicrobial).

Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies., A computationally designed epitope-focused immunogen presenting a single neutralization epitope from Respiratory Syncytial Virus elicits superior epitope-specific responses compared to the viral fusion protein. Furthermore, epitope-focused immunogens can reshape established antibody hierarchies., Author summary Vaccines are one of the most valuable instruments to prevent and control infectious diseases. Their primary correlate of protection is the level of induction of neutralizing antibodies that target critical antigenic sites and thereby block infection. Natural infections with pathogens such as the respiratory syncytial virus (RSV) or influenza induce a broad repertoire of antibodies that target multiple epitopes. Among those, functional antibodies with key specificities are often subdominant (present in low titers). Thus, a central goal for vaccine development is to focus antibody responses on such neutralization epitopes. Here, we show that a computationally designed, epitope-focused immunogen mimicking an important RSV neutralization epitope (site II) can focus antibodies onto this well-defined epitope. In a scenario of preexisting immunity, in which site II–specific antibodies were subdominant, the epitope-focused immunogen selectively boosted site II–specific antibodies, resulting in an increased viral neutralization through this epitope. We propose that rationally designed immunogens spotlighting defined epitopes have a unique potential to focus antibody responses on functionally conserved sites in cases of preexisting immunity. Our results have broad implications for vaccine design as a strategy to steer preexisting antibody responses away from immunodominant, variable epitopes and toward subdominant epitopes that confer broad and potent neutralization.

Picornaviruses are small, icosahedral, nonenveloped, positive-sense, single-stranded RNA viruses that form one of the largest and most important viral families. Numerous Picornaviridae members pose serious health or agricultural threats, causing diseases such as poliomyelitis, hepatitis A, or foot-and-mouth disease. The antigenic characterization of picornavirus capsids plays an important role in understanding the mechanism of viral neutralization and the conformational changes associated with genome release, and it can point to regions which can be targeted by small-molecule compounds to be developed as antiviral inhibitors. In a recent study, Cao and colleagues applied this strategy to hepatitis A virus (HAV) and used cryo-electron microscopy (cryo-EM) to characterize a well-conserved antigenic site recognized by several monoclonal antibodies. They further used computational approaches to identify a small-molecule drug with a strong inhibitory effect on cell attachment., This Primer explores the implications of a recent structural characterization of picornavirus capsid antigenicity, which points to regions that can be targeted by small-molecule antiviral inhibitors.

Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo–electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein–protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation., Tiny pathogenic eukaryotes called microsporidia have evolved highly compact ribosomes, smaller than bacterial ribosomes, and employ diverse hibernation factors to facilitate ribosome inactivation and recovery from the latent spore stage, including the conserved eukaryotic hibernation and recycling factor Lso2.

Determining the duration of protective immunity requires quantifying the magnitude and rate of loss of antibodies to different virus and vaccine antigens. A key complication is heterogeneity in both the magnitude and decay rate of responses of different individuals to a given vaccine, as well as of a given individual to different vaccines. We analyzed longitudinal data on antibody titers in 45 individuals to characterize the extent of this heterogeneity and used models to determine how it affected the longevity of protective immunity to measles, rubella, vaccinia, tetanus, and diphtheria. Our analysis showed that the magnitude of responses in different individuals varied between 12- and 200-fold (95% coverage) depending on the antigen. Heterogeneity in the magnitude and decay rate contribute comparably to variation in the longevity of protective immunity between different individuals. We found that some individuals have, on average, slightly longer-lasting memory than others—on average, they have higher antibody levels with slower decay rates. We identified different patterns for the loss of protective levels of antibodies to different vaccine and virus antigens. Specifically, we found that for the first 25 to 50 years, virtually all individuals have protective antibody titers against diphtheria and tetanus, respectively, but about 10% of the population subsequently lose protective immunity per decade. In contrast, at the outset, not all individuals had protective titers against measles, rubella, and vaccinia. However, these antibody titers wane much more slowly, with a loss of protective immunity in only 1% to 3% of the population per decade. Our results highlight the importance of long-term longitudinal studies for estimating the duration of protective immunity and suggest both how vaccines might be improved and how boosting schedules might be reevaluated., Author summary Immunological memory, mediated by antibodies, is a hallmark of immunity. A key problem for determining the longevity of protective immunity is heterogeneity in the responses of different individuals. We characterize the extent of this heterogeneity and determine how it affects the longevity of protection. We found that some individuals have higher antibody titers and these same individuals tend to have slower decay rates than others. We also found substantial heterogeneity in both the magnitude and decay rate of responses. Furthermore, differences in these two factors contribute comparably to the variation in antibody titers between different individuals over their lifetime. We then use statistical models to determine how variation in the magnitude and decay rate affect how protective immunity is lost at the population level to different virus and vaccine antigens. We identified different patterns for the loss of protective immunity elicited by protein immunization (tetanus and diphtheria) versus replicating viruses (measles, rubella, and vaccinia). While our results agree with the conventional view that antibodies elicited by protein immunization decay faster than those elicited by replicating viruses, we found that this is compensated for by the higher magnitude of responses (relative to the level for protection) for tetanus and diphtheria. Indeed, for the first 4 decades, a higher fraction of vaccinated individuals have protective immunity to tetanus and diphtheria than to measles, rubella, and vaccinia.

Periplasmic flagella are essential for the distinct morphology and motility of spirochetes. A flagella-specific type III secretion system (fT3SS) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella. Here, we deployed cryo-electron tomography (cryo-ET) to visualize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi. We show, for the first time, that the cytosolic ATPase complex is attached to the flagellar C-ring through multiple spokes to form the “spoke and hub” structure in B. burgdorferi. This structure not only strengthens structural rigidity of the round-shaped C-ring but also appears to rotate with the C-ring. Our studies provide structural insights into the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the basis for the development of novel therapeutic strategies against several pathogenic spirochetes., Cryo-electron tomography of periplasmic flagella in the Lyme disease bacterium Borrelia burgdorferi reveals it to have a distinct cytoplasmic ATPase complex and an atypical interaction with the flagellar C-ring., Author summary Type III secretion systems are widely utilized by gram-negative bacteria to assemble flagella or to transport virulence effectors into eukaryotic cells. The central component is known as a type III secretion machine, which consists of a membrane-bound export apparatus and a cytosolic ATPase complex. Powered by the proton motive force and ATP hydrolysis, the secretion machine is responsible for substrate recognition and export. Here, we use the Lyme disease spirochete B. burgdorferi as a model system to unveil unprecedented structural details of the intact flagellar secretion machine by high-throughput cryo-electron tomography (cryo-ET) and subtomogram averaging. We provide the first structural evidence that the cytosolic ATPase complex is attached to the flagellar C-ring through multiple spokes to form the “spoke and hub” structure in B. burgdorferi. The novel architecture of the ATPase complex not only strengthens the flagellar C-ring but also enables an optimal translocation of substrates through the ATPase complex and the export apparatus.

During the aging process, bone marrow mesenchymal stem cells (BMSCs) exhibit declined osteogenesis accompanied by excess adipogenesis, which will lead to osteoporosis. Here, we report that the H3 lysine 36 trimethylation (H3K36me3), catalyzed by histone methyltransferase SET-domain-containing 2 (SETD2), regulates lineage commitment of BMSCs. Deletion of Setd2 in mouse bone marrow mesenchymal stem cells (mBMSCs), through conditional Cre expression driven by Prx1 promoter, resulted in bone loss and marrow adiposity. Loss of Setd2 in BMSCs in vitro facilitated differentiation propensity to adipocytes rather than to osteoblasts. Through conjoint analysis of RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data, we identified a SETD2 functional target gene, Lbp, on which H3K36me3 was enriched, and its expression was affected by Setd2 deficiency. Furthermore, overexpression of lipopolysaccharide-binding protein (LBP) could partially rescue the lack of osteogenesis and enhanced adipogenesis resulting from the absence of Setd2 in BMSCs. Further mechanistic studies demonstrated that the trimethylation level of H3K36 could regulate Lbp transcriptional initiation and elongation. These findings suggest that H3K36me3 mediated by SETD2 could regulate the cell fate of mesenchymal stem cells (MSCs) in vitro and in vivo, indicating that the regulation of H3K36me3 level by targeting SETD2 and/or the administration of downstream LBP may represent a potential therapeutic way for new treatment in metabolic bone diseases, such as osteoporosis., Author summary During the aging process, the ability of the mesenchymal stem cells (MSCs) in the bone marrow to generate bone cells declines, and they become more likely to produce fat cells, resulting in brittle, or osteoporotic, bones. Here, we demonstrate that a histone modification—H3 lysine 36 trimethylation (H3K36me3), which is mediated by the enzyme SET-domain-containing 2 (SETD2) and regulates gene expression—plays an essential role in MSC lineage commitment in the bone marrow. We deleted the Setd2 gene in bone marrow mesenchymal stem cells (BMSCs) in mice and found that these mice exhibited reduced bone formation and increased marrow fat accumulation. Notably, we identified Lbp as one of SETD2 functional downstream genes. Overexpression of lipopolysaccharide-binding protein (LBP) could partially rescue the phenotype of SETD2 deficiency in bone marrow stem cells. Further mechanistic studies revealed that H3K36me3 could regulate Lbp transcription by modulating its transcriptional initiation and elongation. Thus, our study provides a new mechanism for MSC fate determination and a therapeutic target for the treatment of osteoporosis.

The immune state of wild animals is largely unknown. Knowing this and what affects it is important in understanding how infection and disease affects wild animals. The immune state of wild animals is also important in understanding the biology of their pathogens, which is directly relevant to explaining pathogen spillover among species, including to humans. The paucity of knowledge about wild animals' immune state is in stark contrast to our exquisitely detailed understanding of the immunobiology of laboratory animals. Making an immune response is costly, and many factors (such as age, sex, infection status, and body condition) have individually been shown to constrain or promote immune responses. But, whether or not these factors affect immune responses and immune state in wild animals, their relative importance, and how they interact (or do not) are unknown. Here, we have investigated the immune ecology of wild house mice—the same species as the laboratory mouse—as an example of a wild mammal, characterising their adaptive humoral, adaptive cellular, and innate immune state. Firstly, we show how immune variation is structured among mouse populations, finding that there can be extensive immune discordance among neighbouring populations. Secondly, we identify the principal factors that underlie the immunological differences among mice, showing that body condition promotes and age constrains individuals’ immune state, while factors such as microparasite infection and season are comparatively unimportant. By applying a multifactorial analysis to an immune system-wide analysis, our results bring a new and unified understanding of the immunobiology of a wild mammal., Author summary The immune state of wild animals—and the factors that affect this—is largely unknown. Knowing this is important in understanding how infection and disease affects wild animals, and the biology of their pathogens. The paucity of knowledge about wild animals' immune state is in stark contrast to our exquisitely detailed understanding of the immunobiology of laboratory animals. Here, we have investigated the immune ecology of wild house mice—the same species as the laboratory mouse—characterising their adaptive humoral, adaptive cellular, and innate immune state. Firstly, we investigated how immune variation is structured among mouse populations, finding that there can be extensive immune discordance among neighbouring populations. Secondly, we identified the principal factors that underlie the immunological differences among mice, showing that body condition promotes and age constrains individuals’ immune state. By applying a multifactorial analysis to an immune system-wide analysis, our results bring a new and unified understanding of the immunobiology of a wild mammal.

Chronic pain is a debilitating problem, and insights in the neurobiology of chronic pain are needed for the development of novel pain therapies. A genome-wide association study implicated the 5p15.2 region in chronic widespread pain. This region includes the coding region for FAM173B, a functionally uncharacterized protein. We demonstrate here that FAM173B is a mitochondrial lysine methyltransferase that promotes chronic pain. Knockdown and sensory neuron overexpression strategies showed that FAM173B is involved in persistent inflammatory and neuropathic pain via a pathway dependent on its methyltransferase activity. FAM173B methyltransferase activity in sensory neurons hyperpolarized mitochondria and promoted macrophage/microglia activation through a reactive oxygen species–dependent pathway. In summary, we uncover a role for methyltransferase activity of FAM173B in the neurobiology of pain. These results also highlight FAM173B methyltransferase activity as a potential therapeutic target to treat debilitating chronic pain conditions., Author summary Pain is an evolutionarily conserved physiological phenomenon necessary for survival. Yet, pain can become pathological when it occurs independently of noxious stimuli. The molecular mechanisms of pathological pain are still poorly understood, limiting the development of highly needed novel analgesics. Recently, genetic variations in the genomic region encoding FAM173B—a functionally uncharacterized protein—have been linked to chronic pain in humans. In this study, we identify the role and function of FAM173B in the development of pathological pain. We used genetic, biochemical, and behavioral approaches in mice to show that FAM173B is a mitochondrial lysine methyltransferase—a protein that transfers methyl group to donor proteins. By genetically silencing or overexpressing FAM173B in sensory neurons, we showed that FAM173B methyltransferase activity promotes the development of chronic pain. In addition, we discovered that FAM173B methyltransferase activity in the mitochondria of sensory neurons promotes chronic pain via a pathway that depends on the production of reactive oxygen species and on the engagement of spinal cord microglia—engulfing cells of the central nervous system. These data point to an essential role of FAM173B in the regulation of pathological pain.

The bacterium Staphylococcus aureus is an important cause of the life-threatening condition toxic shock syndrome in humans. Bacterial toxins known as superantigens (SAgs) generate this illness by acting as broad activators of a substantial fraction of all T lymphocytes, bypassing the normally highly stringent T-cell receptor antigen specificity to cause a systemic inflammatory cytokine storm in the host. In a new study, Shaler et al. found that immune cells called mucosa-associated invariant T (MAIT) cells make an unexpectedly large contribution to the SAg response in a largely T-cell receptor-independent, cytokine-driven manner. Subsequent to such activation, the MAIT cells remain unresponsive to stimulation with bacterial antigen. Thus, S. aureus hijacks MAIT cells in the cytokine storm and leaves them functionally impaired. This work provides new insight into the role of MAIT cells in antibacterial immunity and opens new avenues of investigation to understand and possibly treat bacterial toxic shock and sepsis.

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a “cytokine storm” with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm’s initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host’s ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ–specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses., Author summary Superantigens (SAgs) are toxins produced by Staphylococcus aureus and Streptococcus pyogenes, microbes that are responsible for a multitude of infectious diseases and conditions. Once released, SAgs activate many immune cells, resulting in a massive inflammatory response that is often followed by a state of immunosuppression, a state that favors opportunistic infections. Using primary human cells as well as wild-type and genetically altered mice, we have now identified a subset of unconventional, innate-like T lymphocytes, called mucosa-associated invariant T (MAIT) cells, as one of the most powerful and quick-acting sources of inflammatory mediators in the aftermath of systemic exposure to SAgs. We also demonstrate that robust activation of MAIT cells by SAgs quickly leads to their exhaustion, and this exhaustion interferes with their ability to participate in antimicrobial host defense and contributes to the immunosuppressive state. Our findings thus define a pathogenic role for MAIT cells during Gram-positive bacterial infections and also uncover a novel mechanism of SAg-mediated immunosuppression. Accordingly, we propose that MAIT cells can be targeted for efficacious treatment of SAg-mediated illnesses.

The envelope glycoproteins (Envs) of HIV-1 continuously evolve in the host by random mutations and recombination events. The resulting diversity of Env variants circulating in the population and their continuing diversification process limit the efficacy of AIDS vaccines. We examined the historic changes in Env sequence and structural features (measured by integrity of epitopes on the Env trimer) in a geographically defined population in the United States. As expected, many Env features were relatively conserved during the 1980s. From this state, some features diversified whereas others remained conserved across the years. We sought to identify “clues” to predict the observed historic diversification patterns. Comparison of viruses that cocirculate in patients at any given time revealed that each feature of Env (sequence or structural) exists at a defined level of variance. The in-host variance of each feature is highly conserved among individuals but can vary between different HIV-1 clades. We designate this property “volatility” and apply it to model evolution of features as a linear diffusion process that progresses with increasing genetic distance. Volatilities of different features are highly correlated with their divergence in longitudinally monitored patients. Volatilities of features also correlate highly with their population-level diversification. Using volatility indices measured from a small number of patient samples, we accurately predict the population diversity that developed for each feature over the course of 30 years. Amino acid variants that evolved at key antigenic sites are also predicted well. Therefore, small “fluctuations” in feature values measured in isolated patient samples accurately describe their potential for population-level diversification. These tools will likely contribute to the design of population-targeted AIDS vaccines by effectively capturing the diversity of currently circulating strains and addressing properties of variants expected to appear in the future., Author summary HIV-1 is the causative agent of the global AIDS pandemic. The envelope glycoproteins (Envs) of HIV-1 constitute a primary target for antibody-based vaccines. However, the diversity of Envs in the population limits the potential efficacy of this approach. Accurate estimates of the range of variants that currently infect patients and those expected to appear in the future will likely contribute to the design of population-targeted immunogens. We found that different properties (features) of Env have different propensities for small “fluctuations” in their values among viruses that infect patients at any given time point. This propensity of each feature for in-host variance, which we designate “volatility”, is conserved among patients. We apply this parameter to model the evolution of features (in patients and population) as a diffusion process driven by their “diffusion coefficients” (volatilities). Using volatilities measured from a few patient samples from the 1980s, we accurately predict properties of viruses that evolved in the population over the course of 30 years. The diffusion-based model described here efficiently captures evolution of phenotypes in biological systems controlled by a dominant random component.

Type III protein-secretion machines are essential for the interactions of many pathogenic or symbiotic bacterial species with their respective eukaryotic hosts. The core component of these machines is the injectisome, a multiprotein complex that mediates the selection of substrates, their passage through the bacterial envelope, and ultimately their delivery into eukaryotic target cells. The injectisome is composed of a large cytoplasmic complex or sorting platform, a multiring base embedded in the bacterial envelope, and a needle-like filament that protrudes several nanometers from the bacterial surface and is capped at its distal end by the tip complex. A characteristic feature of these machines is that their activity is stimulated by contact with target host cells. The sensing of target cells, thought to be mediated by the distal tip of the needle filament, generates an activating signal that must be transduced to the secretion machine by the needle filament. Here, through a multidisciplinary approach, including solid-state NMR (SSNMR) and cryo electron microscopy (cryo-EM) analyses, we have identified critical residues of the needle filament protein of a Salmonella Typhimurium type III secretion system that are involved in the regulation of the activity of the secretion machine. We found that mutations in the needle filament protein result in various specific phenotypes associated with different steps in the type III secretion process. More specifically, these studies reveal an important role for a polymorphic helix of the needle filament protein and the residues that line the lumen of its central channel in the control of type III secretion., Type III secretion machines evolved to inject bacterial effector proteins into eukaryotic cells, becoming active upon bacterial contact with the target cells. Here we provide insight into the mechanism by which the contact signal is transduced to activate the protein secretion machine.

Individuals chronically infected with HIV-1 harbor complex viral populations within their bloodstreams. Recently, it has come to light that when these people infect others, the new infection is typically established by only one or a small number of virions from within this complex viral swarm. An important goal is to characterize the biological properties of HIV-1 virions that seed and exist early in new human infections because these are potentially the only viruses against which a prophylactic HIV-1 vaccine would need to elicit protection. This includes understanding how the Envelope (Env) protein of these virions interacts with the T-cell receptor CD4, which supports attachment and entry of HIV-1 into target cells. We examined early HIV-1 isolates for their ability to infect cells via the CD4 receptor of 15 different primate species. Primates were the original source of HIV-1 and now serve as valuable animal models for studying HIV-1. We find that most primary isolates of HIV-1 from the blood, including early isolates, are highly selective and enter cells through some primate CD4 receptor orthologs but not others. This phenotype is remarkably consistent, regardless of route of transmission, viral subtype, or time of isolation post infection. We show that the weak CD4 binding affinity of blood-derived HIV-1 isolates is what makes them sensitive to the small sequence differences in CD4 from one primate species to the next. To substantiate this, we engineered an early HIV-1 Env to have high, medium, or low binding affinity to CD4, and we show that it loses the ability to enter cells via the CD4 receptor of many primate species as the binding affinity gets weaker. Based on the phenotype of selective use of primate CD4, we find that weak CD4 binding appears to be a nearly universal property of HIV-1 circulating in the bloodstream. Therefore, weak binding to CD4 must be a selected and important property in the biology of HIV-1 in the body. We identify six primate species that encode CD4 receptors that fully support the entry of early HIV-1 isolates despite their low binding affinity for CD4. These findings will help inform long-standing efforts to model HIV-1 transmission and early disease in primates., The current animal model for HIV, the macaque, encodes a CD4 receptor that is non-permissive for HIV entry. This paper reveals that six primate species encode CD4 receptors compatible with HIV infection, potentially making them powerful tools for the study of HIV biology. Furthermore, weak CD4 binding is a nearly constant, and apparently selected, property of HIV circulating in the human bloodstream.

Hepatitis A virus (HAV), an enigmatic and ancient pathogen, is a major causative agent of acute viral hepatitis worldwide. Although there are effective vaccines, antivirals against HAV infection are still required, especially during fulminant hepatitis outbreaks. A more in-depth understanding of the antigenic characteristics of HAV and the mechanisms of neutralization could aid in the development of rationally designed antiviral drugs targeting HAV. In this paper, 4 new antibodies—F4, F6, F7, and F9—are reported that potently neutralize HAV at 50% neutralizing concentration values (neut50) ranging from 0.1 nM to 0.85 nM. High-resolution cryo-electron microscopy (cryo-EM) structures of HAV bound to F4, F6, F7, and F9, together with results of our previous studies on R10 fragment of antigen binding (Fab)-HAV complex, shed light on the locations and nature of the epitopes recognized by the 5 neutralizing monoclonal antibodies (NAbs). All the epitopes locate within the same patch and are highly conserved. The key structure-activity correlates based on the antigenic sites have been established. Based on the structural data of the single conserved antigenic site and key structure-activity correlates, one promising drug candidate named golvatinib was identified by in silico docking studies. Cell-based antiviral assays confirmed that golvatinib is capable of blocking HAV infection effectively with a 50% inhibitory concentration (IC50) of approximately 1 μM. These results suggest that the single conserved antigenic site from complete HAV capsid is a good antiviral target and that golvatinib could function as a lead compound for anti-HAV drug development., Structures of hepatitis A virus in complex with five neutralizing antibodies reveal a single conserved antigenic site and pinpoint key structure-activity correlates, allowing in silico screening to identify a potent candidate inhibitor drug, golvatinib., Author summary Hepatitis A virus (HAV) is a unique, hepatotropic human picornavirus that infects approximately 1.5 million people annually and continues to cause mortality despite a successful vaccine. There are no licensed therapeutic drugs to date. Better knowledge of HAV antigenic features and neutralizing mechanisms will facilitate the development of HAV-targeting antiviral drugs. In this study, we report 4 potent HAV-specific neutralizing monoclonal antibodies (NAbs), together with our previous reported R10, that efficiently inhibit HAV infection by blocking attachment to the host cell. All 5 epitopes are located within the same patch and are highly conserved across 6 genotypes of human HAV, which suggests a single antigenic site for HAV, highlighting a prime target for structure-based drug design. Analysis of complexes with the 5 NAbs with varying neutralizing activities pinpointed key structure-activity correlates. By using a robust in silico docking method, one promising inhibitor named golvatinib was successfully identified from the DrugBank Database. In vitro assays confirmed its ability to block viral infection and revealed its neutralizing mechanism. Our approach could be useful in the design of effective drugs for picornavirus infections.

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The “breathing” of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously., Cryo-EM reveals a new epitope between influenza virus hemagglutinin head protomers that is only transiently exposed, yet leads to a potent, pan H7 influenza-neutralizing response; this unique mechanism of action represents a potential new opportunity to combat influenza., Author summary Influenza viruses cause severe respiratory infections on a global scale annually. Vaccine efforts are hampered by the virus’s naturally high mutation rate, which results in wide variation between influenza strains of the antigens that are produced and recognized by antibodies, particularly in the surface glycoprotein hemagglutinin (HA). However, broadly neutralizing antibodies (bnAbs) are a class of antibodies that develop during natural infections that are capable of inhibiting infection across multiple strains. In this study, we structurally characterized one such bnAb, H7.5, which targets a unique semioccluded yet highly conserved region on the HA head. We showed, using both negative-stain and high-resolution cryo-electron microscopy (cryoEM), that after a short incubation, H7.5 fragment antigen binding (Fab) induces HA to fall apart, effectively preventing infection. We found that H7.5 binds to an epitope only accessible through transient “breathing” of the HA head, and this observation provides insight into the conformational transitions necessary for viral fusion as well as key information about a unique vaccine target.

Fundamental to biological decision-making is the ability to generate bimodal expression patterns where 2 alternate expression states simultaneously exist. Here, we use a combination of single-cell analysis and mathematical modeling to examine the sources of bimodality in the transcriptional program controlling HIV’s fate decision between active replication and viral latency. We find that the HIV transactivator of transcription (Tat) protein manipulates the intrinsic toggling of HIV’s promoter, the long terminal repeat (LTR), to generate bimodal ON-OFF expression and that transcriptional positive feedback from Tat shifts and expands the regime of LTR bimodality. This result holds for both minimal synthetic viral circuits and full-length virus. Strikingly, computational analysis indicates that the Tat circuit’s noncooperative “nonlatching” feedback architecture is optimized to slow the promoter’s toggling and generate bimodality by stochastic extinction of Tat. In contrast to the standard Poisson model, theory and experiment show that nonlatching positive feedback substantially dampens the inverse noise-mean relationship to maintain stochastic bimodality despite increasing mean expression levels. Given the rapid evolution of HIV, the presence of a circuit optimized to robustly generate bimodal expression appears consistent with the hypothesis that HIV’s decision between active replication and latency provides a viral fitness advantage. More broadly, the results suggest that positive-feedback circuits may have evolved not only for signal amplification but also for robustly generating bimodality by decoupling expression fluctuations (noise) from mean expression levels., Author summary A central and recurring feature of cell-fate regulating circuits is their ability to generate bimodal expression—2 alternate expression states that exist simultaneously—with each state corresponding to a different cell fate. To understand the mechanisms enabling bimodality in a natural decision-making circuit, we examined HIV’s fate-selection circuit, the Tat circuit. This bimodal circuit is sufficient and necessary to generate a bet-hedging decision between 2 alternate HIV fates: active viral replication and long-lived dormancy (proviral latency). The dormant state, which is resistant to most antiviral drugs, is the primary clinical barrier to curing an HIV infection. While the canonical role of positive feedback is to amplify a signal, surprisingly, we find that the HIV transactivator of transcription (Tat) positive-feedback architecture is instead optimized to expand the regime of HIV expression bimodality. From an evolutionary perspective, the results suggest that positive-feedback circuits may have evolved to robustly generate bimodality in certain contexts, and, given the rapid evolution of HIV, the presence of a circuit optimized to robustly generate bimodal expression patterns appears to support the hypothesis that HIV’s active-versus-latent decision confers viral fitness.

In the last 15 years, antiretroviral therapy (ART) has been the most globally impactful life-saving development of medical research. Antiretrovirals (ARVs) are used with great success for both the treatment and prevention of HIV infection. Despite these remarkable advances, this epidemic grows relentlessly worldwide. Over 2.1 million new infections occur each year, two-thirds in women and 240,000 in children. The widespread elimination of HIV will require the development of new, more potent prevention tools. Such efforts are imperative on a global scale. However, it must also be recognised that true containment of the epidemic requires the development and widespread implementation of a scientific advancement that has eluded us to date—a highly effective vaccine. Striving for such medical advances is what is required to achieve the end of AIDS., This essay argues that while the tools to control the HIV epidemic are already at our disposal, to end AIDS we need innovative strategies for HIV vaccine design, microbicide platforms, and an HIV cure.