Infection of enterovirus 71 (EV71) is affected by cell surface receptors, including the scavenger receptor B2, which are required for viral uncoating, and attachment receptors, such are heparan sulfate (HS), which bind virus, but do not support uncoating. Amino acid residue 145 of the capsid protein VP1 affects viral binding to HS, and virulence in mice. However, the contribution of this amino acid to pathogenicity in humans is not known. We produced EV71 having glycine (VP1-145G) or glutamic acid (VP1-145E) at position 145. VP1-145G, but not VP1-145E, enhanced viral infection in cell culture in an HS-dependent manner. However, VP1-145G showed an attenuated phenotype in wild-type suckling mice and in a transgenic mouse model expressing human scavenger receptor B2 (hSCARB2), while VP1-145E virus showed a virulent phenotype in both models. Thus HS-binding property and virulence are negatively correlated. Immunohistochemical analyses showed that HS is highly expressed in vascular endothelial cells and some other cell types where hSCARB2 is expressed at low or undetectable levels. VP1-145G virus bound to tissue homogenate of both hSCARB2 transgenic and non-transgenic mice, and viral titer was reduced in the bloodstream immediately after intravenous inoculation. Furthermore, VP1-145G virus failed to disseminate well in the mouse organs. These data suggest that VP1-145G virus is adsorbed by attachment receptors such as HS, during circulation leading to abortive infection of HS-positive cells. This trapping effect is thought to be a major mechanism of attenuation of the VP1-145G virus. Attachment receptors expressed on the host cell surface are thought to enhance EV71 infection by increasing the chance of encountering true receptors. Although this has been confirmed using cell culture for some viruses, the importance of attachment receptors is unknown. This report provides an unexpected answer to this question. We demonstrated that the VP1-145G virus binds to HS and shows an attenuated phenotype in an hSCARB2-dependent animal infection model. HS is highly expressed in cells that express hSCARB2 at low or undetectable levels. Our data indicate that HS binding directs VP1-145G virus towards abortive infection, and keeps virus away from hSCARB2-positive cells. Thus, although the ability of VP1-145G virus to use HS might be an advantage in replication in certain cultured cells, it becomes a serious disadvantage in replication This adsorption is thought to be a major mechanism of attenuation associated with attachment receptor usage.
Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and sometimes causes severe or fatal neurological complications. The amino acid at VP1-145 determines virological characteristics of EV71. Viruses with glutamic acid (E) at VP1-145 (VP1-145E) are virulent in neonatal mice and transgenic mice expressing human scavenger receptor B2, whereas those with glutamine (Q) or glycine (G) are not. However, the contribution of this variation to pathogenesis in humans is not fully understood. We compared the virulence of VP1-145E and VP1-145G viruses of Isehara and C7/Osaka backgrounds in cynomolgus monkeys. VP1-145E, but not VP1-145G, viruses induced neurological symptoms. VP1-145E viruses were frequently detected in the tissues of infected monkeys. VP1-145G viruses were detected less frequently and disappeared quickly. Instead, mutants that had a G to E mutation at VP1-145 emerged, suggesting that VP1-145E viruses have a replication advantage in the monkeys. This is consistent with our hypothesis proposed in the accompanying paper that the VP1-145G virus is attenuated due to its adsorption by heparan sulfate. Monkeys infected with both viruses produced neutralizing antibodies before the onset of the disease. Interestingly, VP1-145E viruses were more resistant to neutralizing antibodies than VP1-145G viruses A small amount of neutralizing antibody raised in the early phase of infection may not be sufficient to block the dissemination of VP1-145E viruses. The different resistance of the VP1-145 variants to neutralizing antibodies may be one of the reasons for the difference in virulence. The contribution of VP1-145 variants in humans is not fully understood. In some reports, VP1-145G/Q viruses were more frequently isolated from severely affected than from mildly affected patients, suggesting that VP1-145G/Q viruses are more virulent. In the accompanying paper, we showed that VP1-145E viruses are more virulent than VP1-145G viruses in human SCARB2 transgenic mice. Heparan sulfate acts as a decoy to specifically trap the VP1-145G viruses and leads to abortive infection. Here, we demonstrated that VP1-145G was attenuated in cynomolgus monkeys, suggesting that this hypothesis is also true in a non-human primate model. VP1-145E viruses, but not VP1-145G viruses, were highly resistant to neutralizing antibodies. We propose the difference in resistance against neutralizing antibodies as another mechanism of EV71 virulence. In summary, VP1-145 contributes to virulence determination by controlling attachment receptor usage and antibody sensitivity.
Enterovirus 71 (EV-A71) is one of the major causative agents of hand, foot, and mouth disease. EV-A71 infection is sometimes associated with severe neurological diseases such as acute encephalitis, acute flaccid paralysis, and cardiopulmonary failure. Therefore, EV-A71 is a serious public health concern. Scavenger receptor class B, member 2 (SCARB2) is a type III transmembrane protein that belongs to the CD36 family and is a major receptor for EV-A71. SCARB2 supports attachment and internalization of the virus and initiates conformational changes that lead to uncoating of viral RNA in the cytoplasm. The three-dimensional structure of the virus-receptor complex was elucidated by cryo-electron microscopy. Two α-helices in the head domain of SCARB2 bind to the G-H loop of VP1 and the E-F loop of VP2 capsid proteins of EV-A71. Uncoating takes place in a SCARB2-and low pH-dependent manner. In addition to SCARB2, other molecules support cell surface binding of EV-A71. Heparan sulfate proteoglycans, P-selectin glycoprotein ligand-1, sialylated glycan, annexin II, vimentin, fibronectin, and prohibitin enhance viral infection by retaining the virus on the cell surface. These molecules are known as "attachment receptors" because they cannot initiate uncoating. In vivo, SCARB2 expression was observed in EV-A71 antigenpositive neurons and epithelial cells in the crypts of the palatine tonsils in patients that died of EV-A71 infection. Adult mice are not susceptible to infection by EV-A71, but transgenic mice that express human SCARB2 become susceptible to EV-A71 infection and develop neurological diseases similar to those observed in humans. Attachment receptors may also be involved in EV-A71 infection in vivo. Although heparan sulfate proteoglycans are expressed by many cultured cell lines and enhance infection by a subset of EV-A71 strains, they are not expressed by cells that express SCARB2 at high levels in vivo. Thus, heparan sulfate-positive cells merely adsorb the virus and do not contribute to replication or dissemination of the virus in vivo. In addition to these attachment receptors, cyclophilin A and human tryptophanyl aminoacyl-tRNA synthetase act as an uncoating regulator and an entry mediator that can confer susceptibility to non-susceptibile cells in the absence of SCARB2, respectively. The roles of attachment receptors and other molecules in EV-A71 pathogenesis remain to be elucidated.
Toxoplasma gondii CDPK1 (TgCDPK1) was found to be the target of the toxoplasmocidal compound 1NM-PP1. When TgCDPK1 was mutated at position 128 from glycine to methionine, resistance was gained. Inhibition of gliding motility without inhibition of micronemal secretion by 1NM-PP1 suggests a function for TgCDPK1 in gliding motility.
Erythrocyte invasion is critical to the pathogenesis and survival of the malarial parasite, Plasmodium falciparum. This process is partly mediated by proteins that belong to the Duffy binding-like family, which are expressed on the merozoite surface. One of these proteins, BAEBL (also known as EBA-140), is thought to bind to glycophorin C in a sialic acid-dependent manner. In this report, by the binding assay between recombinant BAEBL protein and enzyme-treated erythrocytes, we show that the binding of BAEBL to erythrocytes is mediated primarily by sialic acid and partially through heparan sulfate (HS). Because BAEBL binds to several kinds of HS proteoglycans or purified HS, the BAEBL-HS binding was found to be independent of the HS proteoglycan peptide backbone and the presence of sialic acid moieties. Furthermore, both the sialic acid-and HS-dependent binding were disrupted by the addition of soluble heparin. This inhibition may be the result of binding between BAEBL and heparin. Invasion assays demonstrated that HS-dependent binding was related to the efficiency of merozoite invasion. These results suggest that HS functions as a factor that promotes the binding of BAEBL and merozoite invasion. Moreover, these findings may explain the invasion inhibition mechanisms observed following the addition of heparin and other sulfated glycoconjugates.Malaria caused by Plasmodium falciparum kills approximately 1 million people per year. After entering the human bloodstream, the parasite propagates asexually via repeated cycles of erythrocyte invasion, cell division, and cell rupture. The process by which the parasitic merozoites invade erythrocytes involves the following steps: attachment, apical reorientation, junction formation, and the formation of a protective parasitophorous vacuole (1).The Duffy binding-like (DBL) 2 family is composed of adhesion molecules that are critical for junction formation between the apical end of the merozoite and the erythrocyte surface.Proteins in this family, such as EBA-175 (erythrocyte-binding antigen-175), are homologous to Plasmodium vivax Duffybinding protein. These proteins contain one or more DBL domains, which are composed of conserved cysteine residues and are associated with erythrocyte binding (2). EBA-175 is bound to glycophorin A on the erythrocyte surface in a sialic acid-dependent manner (3). However, erythrocytes that are treated with neuraminidase remain susceptible to the merozoites of some clones (4), indicating the existence of sialic acidindependent invasion pathways. These alternative pathways are thought to be mediated, at least in part, by other members of the DBL family (5).Parasite growth is inhibited by the addition of heparin, some sulfated saccharide anions, and sulfated chemical compounds (6 -11). Although the inhibition mechanisms remain unclear, these reports suggest the possibility that sulfated moieties play a role in merozoite invasion. In addition to invasion, heparan sulfate (HS) is believed to function as a receptor for PfEMP1 (P. falciparum erythr...
Natural abundance of stable nitrogen (N) and oxygen (O) isotopes are invaluable biogeochemical tracers for assessing the N transformations in the environment. To fully exploit these tracers, the N and O isotope effects (15 ε and 18 ε) associated with the respective nitrogen transformation processes must be known. However, the N and O isotope effects of anaerobic ammonium oxidation (anammox), one of the major fixed N sinks and NO 3 − producers, are not well known. Here, we report the dual N and O isotope effects associated with anammox by three different anammox bacteria including "Ca. Scalindua japonica", a putative marine species, which were measured in continuous enrichment culture experiments. All three anammox species yielded similar N isotope effects of NH 4 + oxidation to N 2 (15 ε NH4→N2) ranging from 30.9‰ to 32.7‰ and inverse kinetic isotope effects of NO 2 − oxidation to NO 3 − (15 ε NO2→NO3 = −45.3‰ to −30.1‰). In contrast, 15 ε NO2→N2 (NO 2 − reduction to N 2) were significantly different among three species, which is probably because individual anammox bacteria species might possess different types of nitrite reductase. We also report the combined O isotope effects for NO 2 − oxidation (18 E NO2→NO3) by anammox bacteria. These obtained dual N and O isotopic effects could provide significant insights into the contribution of anammox bacteria to the fixed N loss and NO 2 − reoxidation (N recycling) in various natural environments.
Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is a major cause of morbidity and mortality worldwide. Many candidate genes have been investigated for a possible association with TB. Toll-like receptors (TLRs) are known to play important roles in human innate immune systems. Polymorphisms in and functions of TLRs have been investigated to identify associations with specific infectious diseases, including TB. Here, we examined whether single-nucleotide polymorphisms (SNPs) in TLRs and genes in TLR signaling were associated with TB susceptibility in Indonesian and Vietnamese populations. A statistically significant association was observed between TB susceptibility in a classified Indonesian female group and rs352139, an SNP located in the intron of TLR9, using the genotype (P = 2.76E-04) and recessive (AA vs AG+GG, P = 2.48E-04, odds ratio = 1.827, 95% confidence interval = 1.321-2.526) models. Meta-analysis of the Indonesian and Vietnamese populations showed that rs352139 was significantly associated with TB in the recessive model. This finding indicated that a TLR9 polymorphism might have an important role in the susceptibility to M. tuberculosis in Asian populations.
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