SaeR as a novel target for antivirulence therapy against Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen responsible for a wide range of clinical infections. SaeRS is one of the two-component systems in S. aureus that modulate multiple virulence factors. Although SaeR is required for S. aureus to develop an infection, inhibitors have not been reported. Using an in vivo knockdown method, we demonstrated that SaeR is targetable for the discovery of antivirulence agent. HR3744 was discovered through a high-throughput screening utilizing a GFP-Lux dual reporter system driven by saeP1 promoter. The antivirulence efficacy of HR3744 was tested using Western blot, Quantitative Polymerase Chain Reaction, leucotoxicity, and haemolysis tests. In electrophoresis mobility shift assay, HR3744 inhibited SaeR-DNA probe binding. WaterLOGSY-NMR test showed HR3744 directly interacted with SaeR's DNA-binding domain. When SaeR was deleted, HR3744 lost its antivirulence property, validating the target specificity. Virtual docking and mutagenesis were used to confirm the target's specificity. When Glu159 was changed to Asn, the bacteria developed resistance to HR3744. A structure-activity relationship study revealed that a molecule with a slight modification did not inhibit SaeR, indicating the selectivity of HR3744. Interestingly, we found that SAV13, an analogue of HR3744, was four times more potent than HR3744 and demonstrated identical antivirulence properties and target specificity. In a mouse bacteraemia model, both HR3744 and SAV13 exhibited in vivo effectiveness. Collectively, we identified the first SaeR inhibitor, which exhibited in vitro and in vivo antivirulence properties, and proved that SaeR could be a novel target for developing antivirulence drugs against S. aureus infections.

An immunoassay system to investigate epidemiology of Rocahepevirus ratti (rat hepatitis E virus) infection in humans.

Background & Aims: Rat hepatitis E virus (Rocahepevirus ratti; HEV-C1) is an emerging cause of hepatitis E that is divergent from conventional human-infecting HEV variants (Paslahepevirus balayani; HEV-A). Validated serological assays for HEV-C1 are lacking. We aimed to develop a parallel enzymatic immunoassay (EIA) system that identifies individuals with HEV-C1 exposure. We also aimed to conduct the first HEV-C1 seroprevalence study in humans using this validated EIA system. Methods: Expressed HEV-A (HEV-A4 p239) and HEV-C1 (HEV-C1 p241) peptides were characterised. Blood samples were simultaneously tested in HEV-A4 p239 and HEV-C1 p241 IgG EIAs. An optical density (OD) cut-off-based interpretation algorithm for identifying samples seropositive for HEV-A or HEV-C1 was validated using RT-PCR-positive infection sera. This algorithm was used to measure HEV-C1 seroprevalence in 599 solid organ transplant recipients and 599 age-matched immunocompetent individuals. Results: Both peptides formed virus-like particles. When run in HEV-A4 p239 and HEV-C1 p241 EIAs, HEV-A and HEV-C1 RT-PCR-positive samples formed distinct clusters with minimal overlap in a two-dimensional plot of optical density values. The final EIA interpretation algorithm showed high agreement with RT-PCR results (Cohen's κ = 0.959) and was able to differentiate HEV-A and HEV-C1 infection sera with an accuracy of 94.2% (95% CI: 85.8-98.4%). HEV-C1 IgG seroprevalence was 7/599 (1.2%) among solid organ transplant recipients and 4/599 (0.7%) among immunocompetent individuals. Five of 11 (45.5%) of these patients had history of transient hepatitis of unknown cause. Conclusions: HEV-C1 exposure was identified in 11/1198 (0.92%) individuals in Hong Kong indicating endemic exposure. This is the first estimate of HEV-C1 seroprevalence in humans. The parallel IgG EIA algorithm is a valuable tool for investigating epidemiology and risk factors for HEV-C1 infection. Impact and Implications: Rat hepatitis E virus has recently been discovered to infect humans, but antibody tests for this infection are lacking, making it difficult to gauge how common this infection is. We developed an antibody test algorithm that can identify individuals with past rat hepatitis E virus exposure. We used this algorithm to estimate rat hepatitis E exposure rates in humans in Hong Kong and found that approximately 1% of all tested people had been exposed to this virus previously.

Identification of a Small Molecule Compound Active against Antibiotic-Tolerant Staphylococcus aureus by Boosting ATP Synthesis.

Antibiotic tolerance poses a threat to current antimicrobial armamentarium. Bacteria at a tolerant state survive in the presence of antibiotic treatment and account for persistence, relapse and recalcitrance of infections. Antibiotic treatment failure may occur due to antibiotic tolerance. Persistent infections are difficult to treat and are often associated with poor prognosis, imposing an enormous burden on the healthcare system. Effective strategies targeting antibiotic-tolerant bacteria are therefore highly warranted. In this study, small molecule compound SA-558 was identified to be effective against Staphylococcus aureus that are tolerant to being killed by conventional antibiotics. SA-558 mediated electroneutral transport across the membrane and led to increased ATP and ROS generation, resulting in a reduction of the population of antibiotic-tolerant bacteria. In a murine chronic infection model, of which vancomycin treatment failed, we demonstrated that SA-558 alone and in combination with vancomycin caused significant reduction of MRSA abundance. Our results indicate that SA-558 monotherapy or combinatorial therapy with vancomycin is an option for managing persistent S. aureus bacteremia infection and corroborate that bacterial metabolism is an important target for counteracting antibiotic tolerance.

Linoleic acid metabolism activation in macrophages promotes the clearing of intracellular Staphylococcus aureus.

Multidrug-resistant bacterial pathogens pose an increasing threat to human health. Certain bacteria, such as Staphylococcus aureus, are able to survive within professional phagocytes to escape the bactericidal effects of antibiotics and evade killing by immune cells, potentially leading to chronic or persistent infections. By investigating the macrophage response to S. aureus infection, we may devise a strategy to prime the innate immune system to eliminate the infected bacteria. Here we applied untargeted tandem mass spectrometry to characterize the lipidome alteration in S. aureus infected J774A.1 macrophage cells at multiple time points. Linoleic acid (LA) metabolism and sphingolipid metabolism pathways were found to be two major perturbed pathways upon S. aureus infection. The subsequent validation has shown that sphingolipid metabolism suppression impaired macrophage phagocytosis and enhanced intracellular bacteria survival. Meanwhile LA metabolism activation significantly reduced intracellular S. aureus survival without affecting the phagocytic capacity of the macrophage. Furthermore, exogenous LA treatment also exhibited significant bacterial load reduction in multiple organs in a mouse bacteremia model. Two mechanisms are proposed to be involved in this progress: exogenous LA supplement increases downstream metabolites that partially contribute to LA's capacity of intracellular bacteria-killing and LA induces intracellular reactive oxygen species (ROS) generation through an electron transport chain pathway in multiple immune cell lines, which further increases the capacity of killing intracellular bacteria. Collectively, our findings not only have characterized specific lipid pathways associated with the function of macrophages but also demonstrated that exogenous LA addition may activate lipid modulator-mediated innate immunity as a potential therapy for bacterial infections.

Phosphatidic acid phosphatase 1 impairs SARS-CoV-2 replication by affecting the glycerophospholipid metabolism pathway.

Viruses exploit the host lipid metabolism machinery to achieve efficient replication. We herein characterize the lipids profile reprogramming in vitro and in vivo using liquid chromatography-mass spectrometry-based untargeted lipidomics. The lipidome of SARS-CoV-2-infected Caco-2 cells was markedly different from that of mock-infected samples, with most of the changes involving downregulation of ceramides. In COVID-19 patients' plasma samples, a total of 54 lipids belonging to 12 lipid classes that were significantly perturbed compared to non-infected control subjects' plasma samples were identified. Among these 12 lipid classes, ether-linked phosphatidylcholines, ether-linked phosphatidylethanolamines, phosphatidylcholines, and ceramides were the four most perturbed. Pathway analysis revealed that the glycerophospholipid, sphingolipid, and ether lipid metabolisms pathway were the most significantly perturbed host pathways. Phosphatidic acid phosphatases (PAP) were involved in all three pathways and PAP-1 deficiency significantly suppressed SARS-CoV-2 replication. siRNA knockdown of LPIN2 and LPIN3 resulted in significant reduction of SARS-CoV-2 load. In summary, these findings characterized the host lipidomic changes upon SARS-CoV-2 infection and identified PAP-1 as a potential target for intervention for COVID-19.

Mouse Spexin: (I) NMR Solution Structure, Docking Models for Receptor Binding, and Histological Expression at Tissue Level.

Spexin (SPX), a highly conserved neuropeptide, is known to have diverse functions and has been implicated/associated with pathological conditions, including obesity, diabetes, anorexia nervosa, and anxiety/mood disorders. Although most of the studies on SPX involved the mouse model, the solution structure of mouse SPX, structural aspects for SPX binding with its receptors GalR2/3, and its cellular expression/distribution in mouse tissues are largely unknown. Using CD and NMR spectroscopies, the solution structure of mouse SPX was shown to be in the form of a helical peptide with a random coil from Asn1 to Pro4 in the N-terminal followed by an α-helix from Gln5 to Gln14 in the C-terminus. The molecular surface of mouse SPX is largely hydrophobic with Lys11 as the only charged residue in the α-helix. Based on the NMR structure obtained, docking models of SPX binding with mouse GalR2 and GalR3 were constructed by homology modeling and MD simulation. The models deduced reveal that the amino acids in SPX, especially Asn1, Leu8, and Leu10, could interact with specific residues in ECL1&2 and TMD2&7 of GalR2 and GalR3 by H-bonding/hydrophobic interactions, which provides the structural evidence to support the idea that the two receptors can act as the cognate receptors for SPX. For tissue distribution of SPX, RT-PCR based on 28 tissues/organs harvested from the mouse demonstrated that SPX was ubiquitously expressed at the tissue level with notable signals detected in the brain, GI tract, liver, gonad, and adrenal gland. Using immunohistochemical staining, protein signals of SPX could be located in the liver, pancreas, white adipose tissue, muscle, stomach, kidney, spleen, gonad, adrenal, and hypothalamo-pituitary axis in a cell type-specific manner. Our results, as a whole, not only can provide the structural information for ligand/receptor interaction for SPX but also establish the anatomical basis for our on-going studies to examine the physiological functions of SPX in the mouse model.

CAG RNAs induce DNA damage and apoptosis by silencing NUDT16 expression in polyglutamine degeneration.

DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington's disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.

Differential role of sphingomyelin in influenza virus, rhinovirus and SARS-CoV-2 infection of Calu-3 cells.

Host cell lipids play a pivotal role in the pathogenesis of respiratory virus infection. However, a direct comparison of the lipidomic profile of influenza virus and rhinovirus infections is lacking. In this study, we first compared the lipid profile of influenza virus and rhinovirus infection in a bronchial epithelial cell line. Most lipid features were downregulated for both influenza virus and rhinovirus, especially for the sphingomyelin features. Pathway analysis showed that sphingolipid metabolism was the most perturbed pathway. Functional study showed that bacterial sphingomyelinase suppressed influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, but promoted rhinovirus replication. These findings suggest that sphingomyelin pathway can be a potential target for antiviral therapy, but should be carefully evaluated as it has opposite effects on different respiratory viruses. Furthermore, the differential effect of sphingomyelinase on rhinovirus and influenza virus may explain the interference between rhinovirus and influenza virus infection.

Platinum(II) N-heterocyclic carbene complexes arrest metastatic tumor growth.

Vimentin is a cytoskeletal intermediate filament protein that plays pivotal roles in tumor initiation, progression, and metastasis, and its overexpression in aggressive cancers predicted poor prognosis. Herein described is a highly effective antitumor and antimetastatic metal complex [PtII(C^N^N)(NHC2Bu)]PF6 (Pt1a; HC^N^N = 6-phenyl-2,2'-bipyridine; NHC= N-heterocyclic carbene) that engages vimentin via noncovalent binding interactions with a distinct orthogonal structural scaffold. Pt1a displays vimentin-binding affinity with a dissociation constant of 1.06 µM from surface plasmon resonance measurements and fits into a pocket between the coiled coils of the rod domain of vimentin with multiple hydrophobic interactions. It engages vimentin in cellulo, disrupts vimentin cytoskeleton, reduces vimentin expression in tumors, suppresses xenograft growth and metastasis in different mouse models, and is well tolerated, attributable to biotransformation to less toxic and renal-clearable platinum(II) species. Our studies uncovered the practical therapeutic potential of platinum(II)‒NHC complexes as effective targeted chemotherapy for combating metastatic and cisplatin-resistant cancers.

Clofazimine broadly inhibits coronaviruses including SARS-CoV-2.

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.

Discovery of a Novel Specific Inhibitor Targeting Influenza A Virus Nucleoprotein with Pleiotropic Inhibitory Effects on Various Steps of the Viral Life Cycle.

Influenza A viruses (IAVs) continue to pose an imminent threat to humans due to annual influenza epidemic outbreaks and episodic pandemics with high mortality rates. In this context, the suboptimal vaccine coverage and efficacy, coupled with recurrent events of viral resistance against a very limited antiviral portfolio, emphasize an urgent need for new additional prophylactic and therapeutic options, including new antiviral targets and drugs with new mechanisms of action to prevent and treat influenza virus infection. Here, we characterized a novel influenza A virus nucleoprotein (NP) inhibitor, FA-6005, that inhibited a broad spectrum of human pandemic and seasonal influenza A and B viruses in vitro and protects mice against lethal influenza A virus challenge. The small molecule FA-6005 targeted a conserved NP I41 domain and acted as a potentially broad, multimechanistic anti-influenza virus therapeutic since FA-6005 suppressed influenza virus replication and perturbed intracellular trafficking of viral ribonucleoproteins (vRNPs) from early to late stages. Cocrystal structures of the NP/FA-6005 complex reconciled well with concurrent mutational studies. This study provides the first line of direct evidence suggesting that the newly identified NP I41 pocket is an attractive target for drug development that inhibits multiple functions of NP. Our results also highlight FA-6005 as a promising candidate for further development as an antiviral drug for the treatment of IAV infection and provide chemical-level details for inhibitor optimization.IMPORTANCE Current influenza antivirals have limitations with regard to their effectiveness and the potential emergence of resistance. Therefore, there is an urgent need for broad-spectrum inhibitors to address the considerable challenges posed by the rapid evolution of influenza viruses that limit the effectiveness of vaccines and lead to the emergence of antiviral drug resistance. Here, we identified a novel influenza A virus NP antagonist, FA-6005, with broad-spectrum efficacy against influenza viruses, and our study presents a comprehensive study of the mode of action of FA-6005 with the crystal structure of the compound in complex with NP. The influenza virus inhibitor holds promise as an urgently sought-after therapeutic option offering a mechanism of action complementary to existing antiviral drugs for the treatment of influenza virus infection and should further aid in the development of universal therapeutics.

Clofazimine is a broad-spectrum coronavirus inhibitor that antagonizes SARS-CoV-2 replication in primary human cell culture and hamsters.

COVID-19 pandemic is the third zoonotic coronavirus (CoV) outbreak of the century after severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) since 2012. Treatment options for CoVs are largely lacking. Here, we show that clofazimine, an anti-leprosy drug with a favorable safety and pharmacokinetics profile, possesses pan-coronaviral inhibitory activity, and can antagonize SARS-CoV-2 replication in multiple in vitro systems, including the human embryonic stem cell-derived cardiomyocytes and ex vivo lung cultures. The FDA-approved molecule was found to inhibit multiple steps of viral replication, suggesting multiple underlying antiviral mechanisms. In a hamster model of SARS-CoV-2 pathogenesis, prophylactic or therapeutic administration of clofazimine significantly reduced viral load in the lung and fecal viral shedding, and also prevented cytokine storm associated with viral infection. Additionally, clofazimine exhibited synergy when administered with remdesivir. Since clofazimine is orally bioavailable and has a comparatively low manufacturing cost, it is an attractive clinical candidate for outpatient treatment and remdesivir-based combinatorial therapy for hospitalized COVID-19 patients, particularly in developing countries. Taken together, our data provide evidence that clofazimine may have a role in the control of the current pandemic SARS-CoV-2, endemic MERS-CoV in the Middle East, and, possibly most importantly, emerging CoVs of the future.

Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target.

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.

A broad-spectrum virus- and host-targeting peptide against respiratory viruses including influenza virus and SARS-CoV-2.

The 2019 novel respiratory virus (SARS-CoV-2) causes COVID-19 with rapid global socioeconomic disruptions and disease burden to healthcare. The COVID-19 and previous emerging virus outbreaks highlight the urgent need for broad-spectrum antivirals. Here, we show that a defensin-like peptide P9R exhibited potent antiviral activity against pH-dependent viruses that require endosomal acidification for virus infection, including the enveloped pandemic A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, coronaviruses (SARS-CoV-2, MERS-CoV and SARS-CoV), and the non-enveloped rhinovirus. P9R can significantly protect mice from lethal challenge by A(H1N1)pdm09 virus and shows low possibility to cause drug-resistant virus. Mechanistic studies indicate that the antiviral activity of P9R depends on the direct binding to viruses and the inhibition of virus-host endosomal acidification, which provides a proof of concept that virus-binding alkaline peptides can broadly inhibit pH-dependent viruses. These results suggest that the dual-functional virus- and host-targeting P9R can be a promising candidate for combating pH-dependent respiratory viruses.

Lipidomic Profiling Reveals Significant Perturbations of Intracellular Lipid Homeostasis in Enterovirus-Infected Cells.

Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the most common causes of hand, foot, and mouth disease. Severe EV-A71 and CV-A16 infections may be associated with life-threatening complications. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Lipids are known to play critical roles in multiple stages of the virus replication cycle. The specific lipid profile induced upon virus infection is required for optimal virus replication. The perturbations in the host cell lipidomic profiles upon enterovirus infection have not been fully characterized. To this end, we performed ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS)-based lipidomics to characterize the change in host lipidome upon EV-A71 and CV-A16 infections. Our results revealed that 47 lipids within 11 lipid classes were significantly perturbed after EV-A71 and CV-A16 infection. Four polyunsaturated fatty acids (PUFAs), namely, arachidonic acid (AA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), were consistently upregulated upon EV-A71 and CV-A16 infection. Importantly, exogenously supplying three of these four PUFAs, including AA, DHA, and EPA, in cell cultures significantly reduced EV-A71 and CV-A16 replication. Taken together, our results suggested that enteroviruses might specifically modulate the host lipid pathways for optimal virus replication. Excessive exogenous addition of lipids that disrupted this delicate homeostatic state could prevent efficient viral replication. Precise manipulation of the host lipid profile might be a potential host-targeting antiviral strategy for enterovirus infection.

SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target.

Viruses are obligate intracellular microbes that exploit the host metabolic machineries to meet their biosynthetic demands, making these host pathways potential therapeutic targets. Here, by exploring a lipid library, we show that AM580, a retinoid derivative and RAR-α agonist, is highly potent in interrupting the life cycle of diverse viruses including Middle East respiratory syndrome coronavirus and influenza A virus. Using click chemistry, the overexpressed sterol regulatory element binding protein (SREBP) is shown to interact with AM580, which accounts for its broad-spectrum antiviral activity. Mechanistic studies pinpoint multiple SREBP proteolytic processes and SREBP-regulated lipid biosynthesis pathways, including the downstream viral protein palmitoylation and double-membrane vesicles formation, that are indispensable for virus replication. Collectively, our study identifies a basic lipogenic transactivation event with broad relevance to human viral infections and represents SREBP as a potential target for the development of broad-spectrum antiviral strategies.

Talaromyces marneffei Mp1 Protein, a Novel Virulence Factor, Carries Two Arachidonic Acid-Binding Domains To Suppress Inflammatory Responses in Hosts.

Talaromyces marneffei infection causes talaromycosis (previously known as penicilliosis), a very important opportunistic systematic mycosis in immunocompromised patients. Different virulence mechanisms in T. marneffei have been proposed and investigated. In the sera of patients with talaromycosis, Mp1 protein (Mp1p), a secretory galactomannoprotein antigen with two tandem ligand-binding domains (Mp1p-LBD1 and Mp1p-LBD2), was found to be abundant. Mp1p-LBD2 was reported to possess a hydrophobic cavity to bind copurified palmitic acid (PLM). It was hypothesized that capturing of lipids from human hosts by expressing a large quantity of Mp1p is a virulence mechanism of T. marneffei It was shown that expression of Mp1p enhanced the intracellular survival of T. marneffei by suppressing proinflammatory responses. Mechanistic study of Mp1p-LBD2 suggested that arachidonic acid (AA), a precursor of paracrine signaling molecules for regulation of inflammatory responses, is the major physiological target of Mp1p-LBD2. In this study, we use crystallographic and biochemical techniques to further demonstrate that Mp1p-LBD1, the previously unsolved first lipid binding domain of Mp1p, is also a strong AA-binding domain in Mp1p. These studies on Mp1p-LBD1 support the idea that the highly expressed Mp1p is an effective AA-capturing protein. Each Mp1p can bind up to 4 AA molecules. The crystal structure of Mp1p-LBD1-LBD2 has also been solved, showing that both LBDs are likely to function independently with a flexible linker between them. T. marneffei and potentially other pathogens highly expressing and secreting proteins similar to Mp1p can severely disturb host signaling cascades during proinflammatory responses by reducing the availabilities of important paracrine signaling molecules.

Characterization of the Lipidomic Profile of Human Coronavirus-Infected Cells: Implications for Lipid Metabolism Remodeling upon Coronavirus Replication.

Lipids play numerous indispensable cellular functions and are involved in multiple steps in the replication cycle of viruses. Infections by human-pathogenic coronaviruses result in diverse clinical outcomes, ranging from self-limiting flu-like symptoms to severe pneumonia with extrapulmonary manifestations. Understanding how cellular lipids may modulate the pathogenicity of human-pathogenic coronaviruses remains poor. To this end, we utilized the human coronavirus 229E (HCoV-229E) as a model coronavirus to comprehensively characterize the host cell lipid response upon coronavirus infection with an ultra-high performance liquid chromatography-mass spectrometry (UPLC⁻MS)-based lipidomics approach. Our results revealed that glycerophospholipids and fatty acids (FAs) were significantly elevated in the HCoV-229E-infected cells and the linoleic acid (LA) to arachidonic acid (AA) metabolism axis was markedly perturbed upon HCoV-229E infection. Interestingly, exogenous supplement of LA or AA in HCoV-229E-infected cells significantly suppressed HCoV-229E virus replication. Importantly, the inhibitory effect of LA and AA on virus replication was also conserved for the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV). Taken together, our study demonstrated that host lipid metabolic remodeling was significantly associated with human-pathogenic coronavirus propagation. Our data further suggested that lipid metabolism regulation would be a common and druggable target for coronavirus infections.

Identification and characterization of GLDC as host susceptibility gene to severe influenza.

Glycine decarboxylase (GLDC) was prioritized as a candidate susceptibility gene to severe influenza in humans. The higher expression of GLDC derived from genetic variations may confer a higher risk to H7N9 and severe H1N1 infection. We sought to characterize GLDC as functional susceptibility gene that GLDC may intrinsically regulate antiviral response, thereby impacting viral replication and disease outcome. We demonstrated that GLDC inhibitor AOAA and siRNA depletion boosted IFNß- and IFN-stimulated genes (ISGs) in combination with PolyI:C stimulation. GLDC inhibition and depletion significantly amplified antiviral response of type I IFNs and ISGs upon viral infection and suppressed the replication of H1N1 and H7N9 viruses. Consistently, GLDC overexpression significantly promoted viral replication due to the attenuated antiviral responses. Moreover, GLDC inhibition in H1N1-infected BALB/c mice recapitulated the amplified antiviral response and suppressed viral growth. AOAA provided potent protection to the infected mice from lethal infection, comparable to a standard antiviral against influenza viruses. Collectively, GLDC regulates cellular antiviral response and orchestrates viral growth. GLDC is a functional susceptibility gene to severe influenza in humans.