NanA, a neuraminidase from Streptococcus pneumoniae, shows high levels of sequence diversity, at least in part through recombination with Streptococcus oralis

Streptococcus pneumoniae, an important human pathogen, contains at least two genes, nanA and nanB, that express sialidase activity. NanA is a virulence determinant of pneumococci which is important in animal models of colonization and middle ear infections. The gene encoding NanA was detected in all 106 pneumococcal strains screened that represented 59 restriction profiles. Sequencing confirmed a high level of diversity, up to 17.2% at the nucleotide level and 14.8% at the amino acid level. NanA diversity is due to a number of mechanisms including insertions, point mutations, and recombination generating mosaic genes. The level of nucleotide divergence for each recombinant block is greater than 30% and much higher than the 20% identified within mosaic pbp genes, suggesting that a high selective pressure exists for these alterations. These data indicate that at least one of the four recombinant blocks identified originated from a Streptococcus oralis isolate, demonstrating for the first time that protein virulence determinants of pneumococci have, as identified previously for genes encoding penicillin binding proteins, evolved by recombination with oral streptococci. No amino acid alterations were identified within the aspartic boxes or predicted active site, suggesting that sequence variation may be important in evading the adaptive immune response. Furthermore, this suggests that nanA is an important target of the immune system in the interaction between the pneumococcus and host.     

Structural and functional studies of Streptococcus pneumoniae neuraminidase B: An intramolecular trans-sialidase

The human pathogen Streptococcus pneumoniae expresses neuraminidase proteins that cleave sialic acids from complex carbohydrates. The pneumococcus genome encodes up to three neuraminidase proteins that have been shown to be important virulence factors. Here, we report the first structure of a neuraminidase from S. pneumoniae: the crystal structure of NanB in complex with its reaction product 2,7-anhydro-Neu5Ac. Our structural data, together with biochemical analysis, establish NanB as an intramolecular trans-sialidase with strict specificity towards alpha2-3 linked sialic acid substrates. In addition, we show that NanB differs in its substrate specificity from the other pneumococcal neuraminidase NanA.     

Growth of Streptococcus pneumoniae on human glycoconjugates is dependent upon the sequential activity of bacterial exoglycosidases

In the human host, Streptococcus pneumoniae encounters a variety of glycoconjugates, including mucin, host defense molecules, and glycans associated with the epithelial surface. S. pneumoniae is known to encode a number of glycosidases that may modify these glycoconjugates in vivo. Three exoglycosidases, a neuraminidase (NanA), β-galactosidase (BgaA), and N-acetylglucosaminidase (StrH), have been previously demonstrated to sequentially deglycosylate N-linked glycans on host defense molecules, which coat the pneumococcal surface in vivo. This cleavage is proposed to alter the clearance function of these molecules, allowing pneumococci to persist in the airway. However, we propose that the exoglycosidase-dependent liberation of monosaccharides from these glycoconjugates in close proximity to the pneumococcal surface provides S. pneumoniae with a convenient source of fermentable carbohydrate in vivo. In this study, we demonstrate that S. pneumoniae is able to utilize complex N-linked human glycoconjugates as a sole source of carbon to sustain growth and that efficient growth is dependent upon the sequential deglycosylation of the glycoconjugate substrate by pneumococcal exoglycosidases. In addition to demonstrating a role for NanA, BgaA, and StrH, we have identified a function for the second pneumococcal neuraminidase, NanB, in the deglycosylation of host glycoconjugates and have demonstrated that NanB activity can partially compensate for the loss or dysfunction of NanA. To date, all known functions of pneumococcal neuraminidase have been attributed to NanA. Thus, this study describes the first proposed role for NanB by which it may contribute to S. pneumoniae colonization and pathogenesis.     

Crystal structure of the NanB sialidase from Streptococcus pneumoniae

The Streptococcus pneumoniae genomes encode up to three sialidases (or neuraminidases), NanA, NanB and NanC, which are believed to be involved in removing sialic acid from host cell surface glycans, thereby promoting colonization of the upper respiratory tract. Here, we present the crystal structure of NanB to 1.7 Å resolution derived from a crystal grown in the presence of the buffer Ches (2-N-cyclohexylaminoethanesulfonic acid). Serendipitously, Ches was found bound to NanB at the enzyme active site, and was found to inhibit NanB with a K_i of ∼ 0.5 mM. In addition, we present the structure to 2.4 Å resolution of NanB in complex with the transition-state analogue Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetyl neuraminic acid), which inhibits NanB with a K_i of ∼ 0.3 mM. The sulphonic acid group of Ches and carboxylic acid group of Neu5Ac2en interact with the arginine triad of the active site. The cyclohexyl group of Ches binds in the hydrophobic pocket of NanB occupied by the acetamidomethyl group of Neu5Ac2en. The topology around the NanB active site suggests that the enzyme would have a preference for α2,3-linked sialoglycoconjugates, which is confirmed by a kinetic analysis of substrate binding. NMR studies also confirm this preference and show that, like the leech sialidase, NanB acts as an intramolecular trans-sialidase releasing Neu2,7-anhydro5Ac. All three pneumoccocal sialidases possess a carbohydrate-binding domain that is predicted to bind sialic acid. These studies provide support for a possible differential role for NanB compared to NanA in pneumococcal virulence.     

Sialidase inhibitory activity of diarylnonanoid and neolignan compounds extracted from the seeds of Myristica fragrans

Sialidases are key virulence factors that remove sialic acid from host cell surface glycans, thus unmasking receptors to facilitate bacterial adherence and colonization. In this study, we report the isolation and characterization of novel inhibitors of the Streptococcus pneumoniae sialidases NanA, NanB, and NanC from Myristica fragrans seeds. Of the isolated compounds (1–12), malabaricone C showed the most pneumococcal sialidases inhibition (IC₅₀ of 0.3 μM for NanA, 3.6 μM for NanB, and 2.9 μM for NanC). These results suggested that malabaricone C and neolignans could be potential agents for combating S. pneumoniae infection agents.     

链球菌神经氨酸酶的作用机制及酶活性的测定技术

神经氨酸酶不仅存在于流感病毒,在细菌中也有分布。细菌的神经氨酸酶可裂解宿主体内糖结合物末端的神经氨酸残基,有助于细菌实现在宿主体内的定殖、穿透和扩散,是细菌重要的毒力因子之一。链球菌是自然界广泛存在的人畜共患的病原菌,在多种链球菌中均可检测出神经氨酸酶。肺炎链球菌的神经氨酸酶研究最为透彻,该菌可产生3种神经氨酸酶(NanA,NanB,NanC),NanA不但可以发挥酶的催化作用,分解唾液酸残基,暴露细菌的黏附受体,还能不依赖酶活基团,辅助细菌感染宿主细胞;NanB催化后产物可作为细菌的碳源;NanC可辅助细菌入侵脑部。在无乳链球菌和猪链球菌中,神经氨酸酶的活性一直未得到确切的验证,可能是由于它们的荚膜均含有神经氨酸,所以其神经氨酸酶的活性逐渐在进化中丧失。另外一些链球菌,例如化脓链球菌和C、G、L群链球菌,其神经氨酸酶的底物偏好相近,均对唾液类黏蛋白的催化活性较强,利于链球菌在含唾液类黏蛋白的组织中扩散。在口腔链球菌和血链球菌中,神经氨酸酶破坏血液成分中的神经氨酸链。由此可见,神经氨酸酶的特异性催化作用与链球菌在宿主体内的定植部位密切相关。此外,随着科技的发展,对神经氨酸酶的活性检测,也由早期的硫代巴比妥法,转为现在的荧光值和吸光度的测定,更为便捷和敏感。本文旨在对链球菌的神经氨酸酶的作用机制、与毒力关系及酶活测定方法等研究进展作一综述,为从事相关研究的科学工作者提供参考。     

Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity

An antibacterial approximately 11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5-6.2) and at low temperature (4-35 degrees C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70 degrees C for 15 min, suggesting relatively high protein structure stability. Sequence-analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme.     

Phase variable desialylation of host proteins that bind to Streptococcus pneumoniae in vivo and protect the airway

Most clinical isolates of Streptococcus pneumoniae consist of heterogeneous populations of at least two colony phenotypes, opaque and transparent, selected for in the bloodstream and nasopharynx, respectively. Microarray analysis revealed 24 orfs that demonstrated differences in expression greater than twofold between variants of independent strains. Twenty-one of these showed increased expression in the transparent variants, including 11 predicted to be involved in sugar metabolism. A single genomic region contains seven of these loci including the gene that encodes the neuraminidase, NanA. In contrast to previous studies, there was no contribution of NanA to adherence of S. pneumoniae to epithelial cells or colonization in an animal model. However, we observed NanA-dependent desialylation of human airway components that bind to the organism and may mediate bacterial clearance. Targets of desialylation included human lactoferrin, secretory component, and IgA2 that were shown to be present on the surface of the pneumococcus in vivo during pneumococcal pneumonia. The efficiency of desialylation was increased in the transparent variants and enhanced for host proteins binding to the surface of S. pneumoniae. Because deglycosylation affects the function of many host proteins, NanA may contribute to a protease-independent mechanism to modify bound targets and facilitate enhanced survival of the bacterium.     

Edwardsiella tarda sialidase: Pathogenicity involvement and vaccine potential

Bacterial sialidases are a group of glycohydrolases that are known to play an important role in invasion of host cells and tissues. In this study, we examined in a model of Japanese flounder (Paralichthys olivaceus) the potential function of NanA, a sialidase from the fish pathogen Edwardsiella tarda. NanA is composed of 670 residues and shares low sequence identities with known bacterial sialidases. In silico analysis indicated that NanA possesses a sialidase domain and an autotransporter domain, the former containing five Asp-boxes, a RIP motif, and the conserved catalytic site of bacterial sialidases. Purified recombinant NanA (rNanA) corresponding to the sialidase domain exhibited glycohydrolase activity against sialic acid substrate in a manner that is pH and temperature dependent. Immunofluorescence microscopy showed binding of anti-rNanA antibodies to E.?tarda, suggesting that NanA was localized on cell surface. Mutation of nanA caused drastic attenuation in the ability of E.?tarda to disseminate into and colonize fish tissues and to induce mortality in infected fish. Likewise, cellular study showed that the nanA mutant was significantly impaired in the infectivity against cultured flounder cells. Immunoprotective analysis showed that rNanA in the form of a subunit vaccine conferred effective protection upon flounder against lethal E.?tarda challenge. rNanA vaccination induced the production of specific serum antibodies, which enhanced complement-mediated bactericidal activity and reduced infection of E.?tarda into flounder cells. Together these results indicate that NanA plays an important role in the pathogenesis of E.?tarda and may be exploited for the control of E.?tarda infection in aquaculture.     

Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene.

Pasteurella haemolytica serotype A1 secretes a glycoprotease which is specific for O-sialoglycoproteins such as glycophorin A. The gene encoding the glycoprotease enzyme has been cloned in the recombinant plasmid pH1, and its nucleotide sequence has been determined. The gene (designated gcp) codes for a protein of 35.2 kDa, and an active enzyme protein of this molecular mass can be observed in Escherichia coli clones carrying pPH1. In vivo labeling of plasmid-encoded proteins in E. coli maxicells demonstrated the expression of a 35-kDa protein from pPH1. The amino-terminal sequence of the heterologously expressed protein corresponds to that predicted from the nucleotide sequence. The glycoprotease is a neutral metalloprotease, and the predicted amino acid sequence of the glycoprotease contains a putative zinc-binding site. The gene shows no significant homology with the genes for other proteases of procaryotic or eucaryotic origin. However, there is substantial homology between gcp and an E. coli gene, orfX, whose product is believed to function in the regulation of macromolecule biosynthesis.     

Expression profile of mouse BWF1, a protein with a BEACH domain, WD40 domain and FYVE domain

We isolated a mouse cDNA encoding a protein that contains a BEACH domain, 5 WD40 repeats and a FYVE domain, which we designated as BWF1. The mRNA is approximately 10 kb in size and encodes a protein consisting of 3508 amino acids with a predicted molecular weight of 385 kDa. BWF1 has 45% homology with the Drosophila protein, blue cheese (BCHS). The BWF1 gene consists of 67 exons, which span 270 kb of genomic sequence, and has been mapped to mouse chromosome 5. Northern blot analysis revealed that it was strongly expressed in the liver, moderately in the kidney and testis, and weakly in the brain of adult mice. During the development of the mouse brain, BWF1 mRNA was abundant on embryonic day (E) 14-16; after birth, the level of BWF1 mRNA expression decreased markedly to reach the adult level at postnatal day 3. In situ hybridization analysis revealed that the expressed BWF1 mRNA was restricted to the marginal region both in E14 and E16 embryonic brain, but became diffuse after birth. Confocal microscopy studies of the epitope-tagged BWF1 protein showed that the protein was a cytoplasmic one.     

A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast genome

The photosystem II reaction center complex, so-called D1-D2-cytochrome b-559 complex, isolated from higher plants contains a new component of about 4.8 kDa [(1988) Plant Cell Physiol. 29, 1233-1239]. The partial amino acid sequence of this component from spinach was determined after release of N-terminal blockage. The determined sequence matched an open reading frame (ORF36) of the chloroplast genome from tobacco and liverwort, which is located downstream from the psbK gene and forms an operon with psbK. The predicted product consists of 36 amino acid residues and has a single membrane-spanning segment. High homology between the tobacco and liverwort genes, and its presence in the reaction center complex suggest an important role for this component in the photosystem II complex. Since this gene corresponds to a part of the formerly designated psbI gene, we propose to revise the definition of psbI as the gene encoding the 4.8 kDa reaction center component.     

Activation of brain endothelium by pneumococcal neuraminidase NanA promotes bacterial internalization

Streptococcus pneumoniae (SPN), the leading cause of meningitis in children and adults worldwide, is associated with an overwhelming host inflammatory response and subsequent brain injury. Here we examine the global response of the blood–brain barrier to SPN infection and the role of neuraminidase A (NanA), an SPN surface anchored protein recently described to promote central nervous system tropism. Microarray analysis of human brain microvascular endothelial cells (hBMEC) during infection with SPN or an isogenic NanA‐deficient (ΔnanA) mutant revealed differentially activated genes, including neutrophil chemoattractants IL‐8, CXCL‐1, CXCL‐2. Studies using bacterial mutants, purified recombinant NanA proteins and in vivo neutrophil chemotaxis assays indicated that pneumococcal NanA is necessary and sufficient to activate host chemokine expression and neutrophil recruitment during infection. Chemokine induction was mapped to the NanA N‐terminal lectin‐binding domain with a limited contribution of the sialidase catalytic activity, and was not dependent on the invasive capability of the organism. Furthermore, pretreatment of hBMEC with recombinant NanA protein significantly increased bacterial invasion, suggesting that NanA‐mediated activation of hBMEC is a prerequisite for efficient SPN invasion. These findings were corroborated in an acute murine infection model where we observed less inflammatory infiltrate and decreased chemokine expression following infection with the ΔnanA mutant.     

Deglycosylation of human glycoconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae

Streptococcus pneumoniae produces three surface-associated exoglycosidases; a neuraminidase, NanA, a beta-galactosidase, BgaA, and a beta-N-acetylglucosaminidase, StrH. the proposed functions of NanA, which removes terminal sialic acid, include revealing receptors for adherence, affecting the function of glycosylated host clearance molecules, modifying the surface of other bacteria coinhabiting the same niche, and providing a nutrient source. However, it is unclear whether following desialylation S. pneumoniae can further deglycosylate human targets through the activity of BgaA or StrH. We demonstrate that NanA, BgaA and StrH act sequentially to remove sialic acid, galactose and N-acetylglucosamine and expose mannose on human glycoproteins that bind to the pneumococcus and protect the airway. In addition, both BgaA and NanA were shown to contribute to the adherence of unencapsulated pneumococci, to human epithelial cells. Despite these findings, triple exoglycosidase mutants colonized mice as well as their parental strains, suggesting that any effect of these genes on colonization and disease may be host species-specific. These studies highlight the importance of considering the complete ability of S. pneumoniae to deglycosylate human targets and suggest that in addition to NanA, BgaA and StrH also contribute to pneumococcal colonization and/or pathogenesis.     

Cloning and expression of the pneumococcal neuraminidase gene in Escherichia coli

A gene bank of Sau3AI-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. One clone capable of cleaving the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid was isolated. This activity was inhibited by treatment with a mouse antiserum raised against purified pneumococcal neuraminidase. The recombinant plasmid purified from this clone (designated pJCP301) contained approximately 3.0 kb of pneumococcal DNA. Western-blot analysis indicated that E. coli K-12[pJCP301] produced a 98-kDa polypeptide which reacted with antineuraminidase serum.