The structure of a catalytically active subdomain of the NanA sialidase from S. pneumoniae is reported to a resolution of 2.5 Å. The complex with the inhibitor Neu5Ac2en identifies the key catalytic residues and provides a platform for structure-based development of specific inhibitors.
Streptococcus pneumoniae genomes encode three sialidases, NanA, NanB and NanC, which are key virulence factors that remove sialic acids from various glycoconjugates. The enzymes have potential as drug targets and also as vaccine candidates. The 115 kDa NanA is the largest of the three sialidases and is anchored to the bacterial membrane. Although recombinantly expressed full-length NanA was soluble, it failed to crystallize; therefore, a 56.5 kDa domain that retained full enzyme activity was subcloned. The purified enzyme was crystallized in 0.1 M MES pH 6.5, 30%(w/v) PEG 4000 using the sitting-drop vapour-diffusion method. Data were collected at 100 K to 2.5 Å resolution from a crystal grown in the presence of the inhibitor 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid. The crystal belongs to space group P212121, with unit-cell parameters a = 49.2, b = 95.6, c = 226.6 Å. The structure was solved by molecular replacement and refined to final R and R
free factors of 0.246 and 0.298, respectively.
NanA; sialidases; Streptococcus pneumoniae
Streptococcus pneumoniae is believed to produce more than one form of neuraminidase, but there has been uncertainty as to whether this is due to posttranslational modification of a single gene product or the existence of more than one neuraminidase-encoding gene. Only one stable pneumococcal neuraminidase gene (designated nanA) has been described. In the present study, we isolated and characterized a second neuraminidase gene (designated nanB), which is located close to nanA on the pneumococcal chromosome (approximately 4.5kb downstream). nanB was located on an operon separate from that of nanA, which includes at least five other open reading frames. NanB has a predicted size of 74.5 kDa after cleavage of a 29-amino-acid signal peptide. There was negligible amino acid homology between NanA and NanB, but NanB did exhibit limited homology with the sialidase of Clostridium septicum. NanB was purified from recombinant Escherichia coli and found to have a pH optimum of 4.5, compared with 6.5 to 7.0 for NanA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggested that NanB has a molecular size of approximately 65 kDa. The discrepancy between this estimate and the size predicted from the nucleotide sequence is most likely a consequence of C-terminal processing or anomalous electrophoretic behavior.
Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action.
Clostridium perfringens type D strains cause enteritis and enterotoxemias in livestock after colonizing the intestines and then producing toxins, notably epsilon toxin (ETX). Initially produced and secreted in an inactive form, ETX can be rapidly proteolytically-activated by trypsin and other intestinal proteases. While most C. perfringens strains produce three sialidases, no pathogenic role has yet been identified for these enzymes that remove terminal sialic acid residues from glycoproteins and glycolipids. Our current study found that trypsin increases the activity of the NanI sialidase made by type D strain CN3718. This effect enhanced the ability of NanI to modify the surface of MDCK cells, leading to increased ETX binding and cytotoxicity. We also found that modification of the host cell surface by NanI sialidase allows efficient attachment of CN3718 cells to Caco-2 cells. These results identify interactions between intestinal proteases, ETX, sialidases, and ETX-producing bacteria, whereby trypsin activates not only ETX but also NanI sialidase. If similar effects occur in the intestines, the activated NanI sialidase may modify the host cell surface to facilitate bacterial attachment and thereby worsen disease by facilitating intestinal colonization by type D strains to prolong toxin delivery and, in some species, increase ETX binding.
Sialic acid aldolases or N-acetylneuraminate lyases (NanAs) catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-D-mannosamine (ManNAc). A capillary electrophoresis (CE) assay was developed to directly characterize the activities of NanAs in both Neu5Ac cleavage and Neu5Ac synthesis directions. The assay was used to obtain the pH profile and the kinetic data of a NanA cloned from Pasteurella multocida P-1059 (PmNanA) and a previously reported recombinant Escherichia coli K12 NanA (EcNanA). Both enzymes are active in a broad pH range of 6.0–9.0 in both reaction directions and have similar kinetic parameters. Substrates specificity studies showed that 5-O-methyl-ManNAc, a ManNAc derivative, can be used efficiently as a substrate by PmNanA, but not efficiently by EcNanA, for the synthesis of 8-O-methyl Neu5Ac. In addition, PmNanA (250 mg per liter culture) has a higher expression level (2.5 fold) than EcNanA (94 mg per liter culture). The higher expression level and a broader substrate tolerance make PmNanA a better catalyst than EcNanA for the chemoenzymatic synthesis of sialic acids and their derivatives.
aldolase; capillary electrophoresis; Escherichia coli; lyase; NanA; Pasteurella multocida
Trans-sialidases catalyze the transfer of a sialic acid from one sialoside to an acceptor to form a new sialoside. α2,3-Trans-sialidase activity was initially discovered in the parasitic protozoan Trypanosoma cruzi, and more recently was found in a multifunctional Pasteurella multocida sialyltransferase PmST1. α2,8-Trans-sialidase activity was also described for a multifunctional Campylobacter jejuni sialyltransferase CstII. We report here the discovery of the α2,6-trans-sialidase activity of a previously reported recombinant truncated bacterial α2,6-sialyltransferase from Photobacterium damsela (Δ15Pd2,6ST). This is the first time that the α2,6-trans-sialidase activity has ever been identified. Kinetic studies indicate that Δ15Pd2,6ST-catalyzed trans-sialidase reaction follows a ping-pong bi-bi reaction mechanism. Cytidine 5′-monophosphate, the product of sialyltransferase reactions, is not required by the trans-sialidase activity of the enzyme but enhances the trans-sialidase activity modestly as a non-essential activator. Using chemically synthesized Neu5AcαpNP and LacβMU, α2,6-linked sialoside Neu5Acα2,6LacβMU has been obtained in one-step in high yield using the trans-sialidase activity of Δ15Pd2,6ST. In addition to the α2,6-trans-sialidase activity, Δ15Pd2,6ST also has α2,6-sialidase activity. The multifunctionality is thus a common feature of many bacterial sialyltransferases.
enzyme; sialidase; sialoside; sialyltransferase; trans-sialidase
Cell surface expression of sialic acid has been reported to decrease during immune cell activation, but the significance and regulation of this phenomenon are still being investigated. The major human bacterial pathogen Streptococcus pneumoniae causes pneumonia, sepsis and meningitis, often accompanied by strong inflammatory responses. S. pneumoniae expresses a sialidase (NanA) that contributes to mucosal colonization, platelet clearance, and blood-brain barrier penetration. Using wild-type and isogenic NanA-deficient mutant strains, we showed that S. pneumoniae NanA can desialylate the surface of human THP-1 monocytes, leading to increased ERK phosphorylation, NF-κB activation, and proinflammatory cytokine release. S. pneumoniae NanA expression also stimulates interleukin-8 release and extracellular trap formation from human neutrophils. A mechanistic contribution of unmasking of inhibitory Siglec-5 from cis sialic acid interactions to the proinflammatory effect of NanA is suggested by decreased SHP-2 recruitment to the Siglec-5 intracellular domain and RNA interference studies. Finally, NanA increased production of proinflammatory cytokines in a murine intranasal challenge model of S. pneumoniae pneumonia.
Importance Sialic acids decorate the surface of all mammalian cells and play important roles in physiology, development, and evolution. Siglecs are sialic acid-binding receptors on the surface of immune cells, many of which engage in cis interactions with host sialoglycan ligands and dampen inflammatory responses through transduction of inhibitory signals. Recently, certain bacterial pathogens have been shown to suppress leukocyte innate immune responses by molecular mimicry of host sialic acid structures and engagement of inhibitory Siglecs. Our present work shows that the converse can be true, i.e., that a microbial sialic acid-cleaving enzyme can induce proinflammatory responses, which are in part mediated by unmasking of an inhibitory Siglec. We conclude that host leukocytes are poised to detect and respond to microbial sialidase activity with exaggerated inflammatory responses, which could be beneficial or detrimental to the host depending on the site, stage and magnitude of infection.
Sialic acids decorate the surface of all mammalian cells and play important roles in physiology, development, and evolution. Siglecs are sialic acid-binding receptors on the surface of immune cells, many of which engage in cis interactions with host sialoglycan ligands and dampen inflammatory responses through transduction of inhibitory signals. Recently, certain bacterial pathogens have been shown to suppress leukocyte innate immune responses by molecular mimicry of host sialic acid structures and engagement of inhibitory Siglecs. Our present work shows that the converse can be true, i.e., that a microbial sialic acid-cleaving enzyme can induce proinflammatory responses, which are in part mediated by unmasking of an inhibitory Siglec. We conclude that host leukocytes are poised to detect and respond to microbial sialidase activity with exaggerated inflammatory responses, which could be beneficial or detrimental to the host depending on the site, stage and magnitude of infection.
Clostridium perfringens causes histotoxic infections and diseases originating in animal or human intestines. A prolific toxin producer, this bacterium also produces numerous enzymes, including sialidases, that may facilitate infection. C. perfringens type D strain CN3718 carries genes encoding three sialidases, including two large secreted sialidases (named NanI and NanJ) and one small sialidase (named NanH) that has an intracellular location in log-phase cultures but is present in supernatants of death phase cultures. Using isogenic mutants of CN3718 that are capable of expressing only NanJ, NanI, or NanH, the current study characterized the properties and activities of each sialidase. The optimal temperature determined for NanJ or NanH enzymatic activity was 37°C or 43°C, respectively, while NanI activity increased until temperature reached 48°C. NanI activity was also the most resistant against higher temperatures. All three sialidases showed optimal activities at pH 5.5. Compared to NanJ or NanH, NanI contributed most to the sialidase activity in CN3718 culture supernatants, regardless of the substrate sialic acid linkage; NanI also released the most sialic acid from Caco-2 cells. Only NanI activity was enhanced by trypsin pretreatment and then only for substrates with an α-2,3- or α-2,6-sialic acid linkage. NanJ and NanI activities were more sensitive than NanH activity to two sialidase inhibitors (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid and siastatin B). The activities of the three sialidases were affected differently by several metal ions. These results indicated that each C. perfringens sialidase has distinct properties, which may allow these enzymes to play different roles depending upon environmental conditions.
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.
Sialidases hydrolytically remove sialic acids from sialylated glycoproteins and glycolipids. Sialidases are widely distributed in nature and sialidase-mediated desialylation is implicated in normal and pathological processes. However, mechanisms by which sialidases exert their biological effects remain obscure, in part because sialidase substrate preferences are poorly defined. Here we report the design and implementation of a sialidase substrate specificity assay based on chemoselective labeling of sialosides. We show that this assay identifies components of glycosylated substrates that contribute to sialidase specificity. We demonstrate that specificity of sialidases can depend on structure of the underlying glycan, a characteristic difficult to discern using typical sialidase assays. Moreover, we discovered that S. pneumoniae sialidase NanC strongly prefers sialosides containing the Neu5Ac form of sialic acid, versus those that contain Neu5Gc. We propose using this approach to evaluate sialidase preferences for diverse potential substrates.
Clostridium perfringens can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates. After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and N-acetylmannosamine. The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts N-acetylmannosamine-6-phosphate to N-acetylglucosamine-6-phosphate. A 4.0-kb DNA fragment from C. perfringens NCTC 8798 that contains the nanE and nanA genes has been cloned. The identification of the nanA gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a nanA Escherichia coli strain, EV78. The nanA gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source. By using Northern blot experiments, it was demonstrated that the nanE and nanA genes comprise an operon and that transcription of the operon in C. perfringens is inducible by the addition of sialic acid to the growth medium. The Northern blot experiments also showed that there is no catabolite repression of nanE-nanA transcription by glucose. With a plasmid construct containing a promoterless cpe-gusA gene fusion, in which β-glucuronidase activity indicated that the gusA gene acted as a reporter for transcription, a promoter was localized to the region upstream of the nanE gene. Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the nanE coding sequence.
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.
Streptococcus pneumoniae frequently colonizes the upper respiratory tract of young children and is an important cause of otitis media and invasive disease. Carriage is more common than disease, yet the genetic factors that predispose a given clone for disease are not known. The relationship between capsule type, genetic background, and virulence is complex, and important questions remain regarding how pneumococcal clones differ in their ability to cause disease. Pneumococcal neuraminidase cleaves sialic acid-containing substrates and is thought to be important for pneumococcal virulence. We describe the distribution of multilocus sequence types (ST), capsule type, and neuraminidase genes among 342 carriage, middle ear, blood, and cerebrospinal fluid (CSF) pneumococcal strains from young children. We found 149 STs among our S. pneumoniae isolates. nanA was present in all strains, while nanB and nanC were present in 96% and 51% of isolates, respectively. The distribution of nanC varied among the strain collections from different tissue sources (P = 0.03). The prevalence of nanC was 1.41 (95% confidence interval, 1.11, 1.79) times higher among CSF isolates than among carriage isolates. We identified isolates of the same ST that differed in the presence of nanB and nanC. These studies demonstrate that virulence determinants, other than capsule loci, vary among strains of identical ST. Our studies suggest that the presence of nanC may be important for tissue-specific virulence. Studies that both incorporate MLST and take into account additional virulence determinants will provide a greater understanding of the pneumococcal virulence potential.
Clostridium chauvoei is the causative agent of blackleg, a wide spread serious infection of cattle and sheep with high mortality. In this study we have analyzed the sialidase activity of the NanA protein of C. chauvoei and cloned the sialidase gene nanA. Sialidase is encoded as a precursor protein of 722 amino acids with a 26 amino acid signal peptide. The mature sialidase has a calculated molecular mass of 81 kDa and contains the carbohydrate binding module 32 (CBM32, or F5/8 type C domain), the sialic acid binding module CBM40 and the enzymatically active sialidase domain found in all pro- and eukaryotic sialidases. Sialidase activity does not require the CBM32 domain. The NanA protein is secreted by C. chauvoei as a dimer. The nanA gene was found to be conserved and sialidase activity was found in C. chauvoei strains isolated over a period of 50 years from various geographical locations. Antiserum directed against a recombinant 40 kDa peptide containing CBM40 and part of the enzymatically active domain of NanA neutralized the secreted sialidase activity of all C. chauvoei strains tested.
N-Acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NANA) are good carbon sources for Escherichia coli K-12, whereas N-acetylmannosamine (ManNAc) is metabolized very slowly. The isolation of regulatory mutations which enhanced utilization of ManNAc allowed us to elucidate the pathway of its degradation. ManNAc is transported by the manXYZ-encoded phosphoenolpyruvate-dependent phosphotransferase system (PTS) transporter producing intracellular ManNAc-6-P. This phosphorylated hexosamine is subsequently converted to GlcNAc-6-P, which is further metabolized by the nagBA-encoded deacetylase and deaminase of the GlcNAc-6-P degradation pathway. Two independent mutations are necessary for good growth on ManNAc. One mutation maps to mlc, and mutations in this gene are known to enhance the expression of manXYZ. The second regulatory mutation was mapped to the nanAT operon, which encodes the NANA transporter and NANA lyase. The combined action of the nanAT gene products converts extracellular NANA to intracellular ManNAc. The second regulatory mutation defines an open reading frame (ORF), called yhcK, as the gene for the repressor of the nan operon (nanR). Mutations in the repressor enhance expression of the nanAT genes and, presumably, three distal, previously unidentified genes, yhcJIH. Expression of just one of these downstream ORFs, yhcJ, is necessary for growth on ManNAc in the presence of an mlc mutation. The yhcJ gene appears to encode a ManNAc-6-P-to-GlcNAc-6-P epimerase (nanE). Another putative gene in the nan operon, yhcI, likely encodes ManNAc kinase (nanK), which should phosphorylate the ManNAc liberated from NANA by the NanA protein. Use of NANA as carbon source by E. coli also requires the nagBA gene products. The existence of a ManNAc kinase and epimerase within the nan operon allows us to propose that the pathways for dissimilation of the three amino sugars GlcNAc, ManNAc, and NANA, all converge at the step of GlcNAc-6-P.
Staphylococcus aureus is a ubiquitous bacterial pathogen that is the causative agent of numerous acute and chronic infections. S. aureus colonizes the anterior nares of a significant portion of the healthy adult population, but the mechanisms of colonization remain incompletely defined. Sialic acid (N-acetylneuraminic acid [Neu5Ac]) is a bioavailable carbon and nitrogen source that is abundant on mucosal surfaces and in secretions in the commensal environment. Our findings demonstrate that Neu5Ac can serve as an S. aureus carbon source, and we have identified a previously uncharacterized chromosomal locus (nan) that is required for Neu5Ac utilization. Molecular characterization of the nan locus indicates that it contains five genes, organized into four transcripts, and the genes were renamed nanE, nanR, nanK, nanA, and nanT. Initial studies with gene deletions indicate that nanT, predicted to encode the Neu5Ac transporter, and nanA and nanE, predicted to encode catabolic enzymes, are essential for growth on Neu5Ac. Furthermore, a nanE deletion mutant exhibits a growth inhibition phenotype in the presence of Neu5Ac. Transcriptional fusions and Northern blot analyses indicate that NanR represses the expression of both the nanAT and nanE transcripts, which can be relieved with Neu5Ac. Electrophoretic mobility studies demonstrate that NanR binds to the nanAT and nanE promoter regions, and the Neu5Ac catabolic intermediate N-acetylmannosamine-6-phosphate (ManNAc-6P) relieves NanR promoter binding. Taken together, these data indicate that the nan gene cluster is essential for Neu5Ac utilization and may perform an important function for S. aureus survival in the host.
Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of P. multocida produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced a sialidase gene, nanH, from a fowl cholera isolate of P. multocida. Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases. Insertional inactivation of nanH resulted in a mutant strain that was not deficient in sialidase production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3′ sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3′ and 2-6′ sialyl lactose, while NanH demonstrated activity only on 2-3′ sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8′ sialyl lactose). Recombinant E. coli containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of P. multocida to colonize and persist on vertebrate mucosal surfaces.
Pasteurella multocida is a mucosal pathogen that colonizes the upper respiratory system of rabbits. Respiratory infections can result, but the bacteria can also invade the circulatory system, producing abscesses or septicemia. P. multocida produces extracellular sialidase activity, which is believed to augment colonization of the respiratory tract and the production of lesions in an active infection. Previously, it was demonstrated that some isolates of P. multocida contain two unique sialidase genes, nanH and nanB, that encode enzymes with different substrate specificities (S. Mizan, A. D. Henk, A. Stallings, M. Meier, J. J. Maurer, and M. D. Lee, J. Bacteriol. 182:6874-6883, 2000). We developed a recombinant antigen enzyme-linked immunosorbent assay (ELISA) based on the NanH sialidase of P. multocida and demonstrated that rabbits that were experimentally colonized with P. multocida produce detectable anti-NanH immunoglobulin M (IgM) and IgG in serum, although they demonstrated no clinical signs of pasteurellosis. In addition, clinically ill pet rabbits infected with P. multocida possessed IgM and/or IgG antibody against NanH. The NanH ELISA may be useful for the diagnosis of P. multocida infections in sick rabbits as well as for screening for carriers in research rabbit colonies.
We explored the genetic basis for intraspecific variation in mycoplasmal sialidase activity that correlates with virulence, and its potentially advantageous linkage to nutrient catabolism. Polymorphism in N-acetylneuraminate scavenging and degradation genes (sialidase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate epimerase, N-acetylglucosamine-6-phosphate deacetylase, and glucosamine-6-phosphate deaminase) was evident among eight strains of the avian pathogen Mycoplasma synoviae. Most differences were single nucleotide polymorphisms, ranging from 0.34 ± 0.04 substitutions per 100 bp for N-acetylmannosamine kinase to 0.65 ± 0.03 for the single-copy sialidase gene nanI. Missense mutations were twice as common as silent mutations in nanI; 26% resulted in amino acids dissimilar to consensus; and there was a 12-base deletion near the nanI promoter in strain WVU1853T, supporting a complex genetic basis for differences in sialidase activity. Two strains had identical frameshifts in the N-acetylneuraminate lyase gene nanA, resulting in nonsense mutations, and both had downstream deletions in nanA. Such genetic lesions uncouple extracellular liberation of sialic acid from generation of fructose-6-phosphate and pyruvate via intracellular N-acetylneuraminate degradation. Retention of nanI by such strains, but not others in the M. synoviae phylogenetic cluster, is evidence that sialidase has an important non-nutritional role in the ecology of M. synoviae and certain other mycoplasmas.
Mycoplasma synoviae; polymorphism; N-acetylneuraminate catabolism; sialidase; virulence
The essential toxin in Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis is alpha-toxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ, encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI, nanJ, and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence.
Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. Low cellular expression level of human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty para-nitrophenol (pNP)-tagged α2–3- or α2–6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.
Carbohydrates; Enzymes; Inhibitors; NEU2; Sialidases
Sialidase activity is a putative virulence factor of the anaerobic periodontal pathogen Tannerella forsythia, but it is uncertain which genes encode this activity. Characterization of a putative sialidase, SiaHI, by others, indicated that this protein alone may not be responsible for all of the sialidase activity. We describe a second sialidase in T. forsythia (TF0035), an orthologue of Bacteroides fragilis NanH, and its expression in Escherichia coli. Sialidase activity of the expressed NanH was confirmed by using 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid as a substrate. Biochemical characterization of the recombinant T. forsythia NanH indicated that it was active over a broad pH range, with optimum activity at pH 5.5. This enzyme has high affinity for 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (Km of 32.9 ± 10.3 μM) and rapidly releases 4-methylumbelliferone (Vmax of 170.8 ± 11.8 nmol of 4-methylumbelliferone min−1 mg of protein−1). E. coli lysates containing recombinant T. forsythia NanH cleave sialic acid from a range of substrates, with a preference for α2-3 glycosidic linkages. The genes adjacent to nanH encode proteins apparently involved in the metabolism of sialic acid, indicating that the NanH sialidase is likely to be involved in nutrient acquisition.
Tannerella forsythia is a key contributor to periodontitis, but little is known of its virulence mechanisms. In this study we have investigated the role of sialic acid in biofilm growth of this periodontal pathogen. Our data show that biofilm growth of T. forsythia is stimulated by sialic acid, glycolyl sialic acid, and sialyllactose, all three of which are common sugar moieties on a range of important host glycoproteins. We have also established that growth on sialyllactose is dependent on the sialidase of T. forsythia since the sialidase inhibitor oseltamivir suppresses growth on sialyllactose. The genome of T. forsythia contains a sialic acid utilization locus, which also encodes a putative inner membrane sialic acid permease (NanT), and we have shown this is functional when it is expressed in Escherichia coli. This genomic locus also contains a putatively novel TonB-dependent outer membrane sialic acid transport system (TF0033-TF0034). In complementation studies using an Escherichia coli strain devoid of its outer membrane sialic acid transporters, the cloning and expression of the TF0033-TF0034 genes enabled an E. coli nanR nanC ompR strain to utilize sialic acid as the sole carbon and energy source. We have thus identified a novel sialic acid uptake system that couples an inner membrane permease with a TonB-dependent outer membrane transporter, and we propose to rename these novel sialic acid uptake genes nanO and nanU, respectively. Taken together, these data indicate that sialic acid is a key growth factor for this little-characterized oral pathogen and may be key to its physiology in vivo.
NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments. We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM KCl to 3 M KCl), (2) anion over cation selectivity (Vreversal = +16 mV in 250 mM KCl || 1 M KCl), and (3) two forms of voltage-dependent gating (channel closures above ±200 mV). Single-channel conductance decreases by 50% when HEPES concentration is increased from 100 μM to 100 mM in 250 mM KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers, we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate concentration is increased from 0 mM to 5 mM in 250 mM KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes are on the far side of agar–KCl bridges. A suitable baseline solution for NanC is 250 mM KCl adjusted to pH 7.0 without buffer.
NanC; Single channel; E. coli outer membrane protein; Sialic acid; N-acetylneuraminic acid
Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. α-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, α-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor). Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens.
Sialidase activity varies widely among strains and tends to correlate with strain virulence in the avian pathogen Mycoplasma synoviae. To characterize the forms of selection acting on enzymes required for sialic acid scavenging and catabolism, the ratios of nonsynonymous (Ka) to synonymous (Ks) mutation frequency were calculated for codons in the sialidase gene of 16 strains of M. synoviae and for its nearly identical homolog in four strains of Mycoplasma gallisepticum. The Ka/Ks (ω) values for the linked genes required for nutritive N-acetylneuraminate catabolism (nanA, nagC, nanE, nagA, and nagB) from nine strains of M. synoviae were also determined. To provide context, ω was determined for all corresponding genes of 26 strains of Clostridium perfringens and Streptococcus pneumoniae. Bayesian models of sequence evolution showed that only the sialidase of M. synoviae was under significant (P < 0.001) diversifying selection, while the M. synoviae genes for N-acetylneuraminate catabolism and all genes examined from M. gallisepticum, C. perfringens, and S. pneumoniae were under neutral to stabilizing selection. Diversifying selection acting on the sialidase of M. synoviae, but not on the sialidase of M. gallisepticum or the sialidases or other enzymes essential for sialic acid scavenging in other Firmicutes, is evidence that variation in specific activity of the enzyme is perpetuated by a nonnutritive function in M. synoviae that is influenced by the genomic context of the organism.