INTRODUCTION
All cells are decorated on their surfaces with a complex array of oligosaccharides (glycans) that are attached to membrane glycoproteins and glycolipids or which make up the different components of soluble extracellular materials such as mucus and which are involved in the initial interactions of viruses or other pathogens with cells or mucosal surfaces (
1). The chemical diversity of these glycans contributes to their complex functional roles in controlling cell-cell, cell-pathogen, and cell-environment interactions. Linear and branched glycoconjugates of vertebrate cells are often terminated in sialic acids (Sias). Sias have critical roles in maintaining a variety of cell functions due to their abundance as exposed terminal sugars, where they are involved in highly specific and regulated cellular lectin interactions (
2,
3). They have also been exploited by many different pathogens as well as by nonpathogenic microbes for host recognition and attachment, including playing a role as receptors for important disease-causing viruses, bacteria, and parasites (
4,
5).
Sias are nine-carbon α-keto acids that may be produced and displayed in multiple modified forms that differ in expression between organisms, tissues, and even individual cells (
1,
6,
7). In one series of modifications, the most common form,
N-acetylneuraminic acid (Neu5Ac), may be modified with acetyl groups (O-linked) at carbons -4, -7, -8, and/or -9 (
8,
9). The presence and levels of O-acetylation likely result from combined functions of sialic acid O-acetyl transferases (SOAT) and esterases (SOAE), some of which have been identified and functionally demonstrated, while others are not yet characterized (
10–12). A variety of studies have contributed to our understanding of the relative abundances and distributions of O-acetylated Sias in various eukaryotic systems, as reviewed by Mandal et al. (
13). However, the recent development of recombinant sialoglycan-recognizing probes (SGRPs) recognizing different O-acetylated Sia forms allows their display at the cellular and tissue levels to be analyzed in more detail (
14).
Sia-lectin interactions are critical within organisms, and O-acetyl modifications modulate a variety of intrinsic host functions. Our understanding is clearest for the 9-O-acetyl-modified Sias, which have been detected in human and some other animal tissues with influenza C virus hemagglutinin-esterase fusion (HEF) proteins (
8) and subsequently with porcine
Torovirus HE (
14). Those studies showed that 9-O-acetyl (9-O-Ac) Sias were common in many human tissues and on many cell lines of human origins, as well as being expressed by other animals and displayed within their tissues with highly variable patterns. Removal of 9-O-Ac Sias by expression of the influenza C 9-O-acetylesterase in transgenic mice resulted in developmental arrest of embryos around the 2-cell stage (
15). Regulated 9-O-acetylation is also widely involved in various immunological pathways. For example, modification of the ganglioside GD3 (CD60) of proliferating T cells affects apoptotic pathways and their differentiation states (
16), while Sia 9-O-acetylation on B-cell surface glycans blocks recognition by Sia-binding immunoglobulin-type lectin Siglec-2 (CD22), inhibiting receptor signaling and B-cell activation (
17). Given their involvement in and regulation of cell signaling and proliferation processes, it is not surprising that abnormally displayed O-acetylated Sias are often oncogenic markers (
18–21). Further additions of O-acetyl groups to the 7 and 8 positions give rise to di- and tri-O-acetylated Sias, respectively.
The display and functions of modified 4-O-Ac Sias in intrinsic cell processes are poorly understood. They have been previously identified using high-performance liquid chromatography (HPLC) analyses in certain tissues, blood proteins, and erythrocytes and within the cells of vertebrates of varied lineages, including horses and donkeys, guinea pigs, mice, and monotremes, and in many lineages of bony fish (
14,
22–26). They appear to be distributed in fewer species than the O-acetyl modifications on the Sia glycerol side chain.
Influenza virus particles are decorated with the major glycoproteins hemagglutinin (HA) (displayed as trimers) and neuraminidase (NA) (displayed as tetramers) (
27). Sias act as functional receptors for influenza virus infection as they are recognized and bound by the HA and are cleaved from cell glycans (and viral glycoproteins) by the sialidase function of NA (
28–30). The specific chemistry and linkages of the cell Sias have long been known to influence HA and NA specificity and, ultimately, influenza virus infectivity. Influenza A virus (IAV) and influenza B virus (IBV) HAs from many hosts show strong preferences for binding and infection using Neu5Ac as a receptor, while modified Sias, including Neu5Gc and O-acetyl forms, may reduce or alter infection by those viruses (
31). The Sia sugar linkage to the penultimate residue (α2-3 versus α2-6) affects HA Sia binding affinity and also contributes to host tropism differences between avian and human influenza virus strains (
32,
33). O-acetyl modified Sias have been reported to affect influenza virus infections by altering the activities of HA and NA. One reported effect of O-acetylation is a general reduction in susceptibility to cleavage by sialidases, including influenza virus NA (
34). A number of inhibitors of influenza viruses were identified historically, including the γ-inhibitors, which are defined as sugar molecules that interact with influenza viruses to inhibit infection by acting as “decoy” receptors (
35–38). The 9-O-Ac Sias were also reported to reduce the sialidase activity of NA by almost 3-fold (
39). Strong inhibition of infection and cell-to-cell spread of some human IAVs by horse and guinea pig sera was associated with the presence of high levels of 4-O-Ac Sias on plasma glycoproteins, including α-2-macroglobulin (
35,
37,
40–43).
In other cases, the O-acetyl-modified Sias are required for cell infection by viruses. Both influenza C viruses and influenza D viruses encode a HEF glycoprotein that binds 9-O-Ac Sias as a functional receptor and that also contains a 9-O-Ac-specific esterase that promotes viral release from that Sia form (
44,
45). The orthomyxovirus salmon anemia virus and the coronavirus mouse hepatitis virus-S strain each use 4-O-Ac Sia as a specific receptor for cell binding and infection and also express esterases that can remove that modification (
46,
47).
The modified Sia may also alter the cell and tissue interactions with other viruses, as well as modifying infections of various bacteria and parasites. For example, reovirus type 3 infection and replication were inhibited by the presence of 9-O-Ac Sia (
48), as was
Plasmodium falciparum binding to and invasion of erythrocytes (
49). Many bacteria—both pathogens and commensals—bind to Sia and often express sialidases (neuramindases) that cleave it from glycans, and those activities are likely susceptible to the effects of modifications of the Sia substrate (
50–53). In the bacterial oral pathogen
Tannerella forsythia, an acetylesterase (NanS) is encoded within the Sia operon to remove human 9-O-Ac modifications and improve NanH sialidase function (
54). Understanding the differences in the display and cell or tissue distributions of these modified Sia forms between various animals would therefore be of general interest for understanding how they may alter animal susceptibility to pathogens. Identifying their patterns of expression in tissues of hosts for influenza virus would also help to clarify their roles in controlling HA and NA specificities and activities and hence their effects on tropisms and host ranges of those viruses (
55).
Here we used previously described recombinant soluble viral SGRPs or “virolectins” (
14) with specificities for different O-acetylated Sias, as well as esterases active against the same modifications, to survey the diversity of these modified forms on the cells and tissues that are targets for infection by influenza virus and other viruses in their natural hosts, as well as in some animal models.
DISCUSSION
The overall goal of this study was to survey for the expression of the different modified forms of Sia on the cells and tissues of animals that are natural or experimental hosts of influenza viruses. We were able to confirm and extend prior work which showed that 4-O-Ac, 9-O-Ac, and 7,9-O-Ac Sias are all widely but very variably distributed in the target tissues of animals that are hosts for influenza virus, on the cells of chicken embryos, and on the cultured cells that are frequently used to propagate influenza viruses
in vitro. In general, 7,9-O-Ac and 9-O-Ac Sias were broadly present across various species and their respective tissues and cell lines, while the 4-O-Ac Sias were widely detected in the examined tissues from some animals (horses and guinea pigs) but were not detected in the tissues from others (humans and pigs). We further established a direct comparison of the distributions of Sia linkages, α2-6 and α2-3, in these tissues using plant lectins SNA and MAH, respectively. Our findings in this study are summarized in
Table 1.
The cell lines examined are commonly used for influenza virus laboratory isolation and growth studies, as are embryonated chicken eggs. Tissues sampled in the initial screening included those that are commonly infected by influenza viruses as well as by other respiratory viruses and bacteria. The respiratory tract tissues examined were those from natural or laboratory animal hosts of influenza viruses, while the intestines of ducks—which are among the natural reservoirs of those viruses—were also examined. The goal of this study was to form an initial picture of the distribution of the modified Sias, some of which have been reported to interfere with receptor binding and sialidase activity, to more clearly reveal their possible impact on viral replication and transmission. The results provide initial data that can be used for the design of studies for examining in detail the effects of the Sia modifications on influenza virus infection and spread.
Display of 4-O-Ac Sias.
The first conclusion was that 4-O-Ac Sias are displayed in the respiratory tract tissues of guinea pigs and horses at high levels, as was likely based on previous reports of the presence of modified Sias on eyrthrocytes and blood proteins from those hosts (
37,
61,
62). We observed some display of 4-O-Ac Sias in mice, but the amount seen in respiratory tissues was not as great. Previous reports (and our own unreported survey) showed that 4-O-Ac Sias in mice are highly tissue regulated and most abundantly found in the gastrointestinal tract (
14). That Sia form was also identified in a very small number of cells within the duck, dog, and ferret respiratory tissues screened but not in the tissues of humans and pigs, and it appears not to be displayed on a variety of tissues examined in other studies in humans (
14). We do not yet know the gene(s) encoding the 4-O-acetyltransferase, so it is unclear whether the trace display or absence of this modified Sia represents a genetic loss of the modifying enzyme in some species or variably regulated expression. The high levels of display of 4-O-Ac Sias in horses and guinea pigs are of interest, as horses have been the natural host of the H7N7 influenza virus, of two different H3N8 equine influenza viruses, and of an unknown influenza strain that spread widely around the year 1872 (
63), while guinea pigs are naturally infected by and can transmit many strains of human influenza virus, including both H1N1 and H3N2 lineages (
64). The presence of 4-O-Ac Sias in the respiratory tract therefore appears not to be a high barrier to at least some influenza virus infections, and it is currently not known whether their presence impacts the functions and evolution of HA and NA within the infecting influenza virus populations. The guinea pig tissues examined here did not display the 4-O-Ac Sia on airway-exposed tissues in the upper respiratory tract (trachea), so its role in laboratory infections performed with human viruses is unknown. Horse and guinea pig sera have long been known to be potent inhibitors of several H2 and H3 human influenza strains (
35,
36,
65), and resistance to those sera could be selected through mutations in the HA at residue 145 (H3) that ablate 4-O-Ac binding (
61). The presence of various amounts of sialidase-resistant Sias may therefore contribute to selection of influenza virus populations with HA variants, while its display on some horse and guinea pig cells in culture may provide an
in vitro system suitable for the study of this modified Sia and its interactions with influenza and other viruses. Little is known about the direct effects of 4-O-Ac Sia on other microbes and pathogens, but their effects on sialidase (neuramindase) function would likely alter susceptibility in many respiratory viral and bacterial pathogens (
66–68).
Display of 9-O-Ac and 7,9-O-Ac Sias.
Both 9-O-Ac and 7,9-O-Ac Sias were widely displayed within the respiratory tissues of the influenza virus hosts, although with various locations and abundance levels. Previous studies performed using the influenza C HEF protein or HE-Fc proteins from bovine coronavirus and porcine
Torovirus as probes for 7,9-O-Ac or 9-O-Ac Sias showed that they were widely displayed with significant variations (
8,
14). The finding that high levels of both 9-O-Ac and 7,9-O-Ac Sias are found in the respiratory tracts of humans suggests that those may be a natural ligand for the human influenza viruses, as does the finding of both those modified Sias on MDCK cells and of the 9-O-Ac Sia in embyonated chicken chorioallantoic membrane (CAM) cells. The 9-O-Ac Sias are the primary receptor for influenza C viruses, so their expression in the respiratory tissues of humans and on chicken egg membranes allows infection by that virus (
69). However, the 9-O-Ac group may block binding of some viruses, including strains of influenza A virus. The presence of 9-O-Ac Sias in the submucosal glands of the human trachea suggests that they would be displayed in mucus as part of the biophysical barrier to influenza virus contacting epithelial cell receptors (
70–73). The animal with the distribution of 9-O-Ac Sias that most closely mirrored that of humans was the mouse. Their specific distribution in the respiratory tracts of other mammalian species was more varied but included display in cells of the tracheal epithelium of pigs, horses, and dogs. The influenza D virus group also utilizes the 9-O-Ac Sia receptor, indicating that this Sia form is present in functional amounts in the respiratory tracts of at least pigs, cows, and other ruminants (
45), and 9-O-acetyl forms are present at high levels in bovine submaxillary mucin (
Fig. 1). In contrast, we were unable to detect 9-O-Ac Sias in the given respiratory tissues (trachea and lung) of guinea pigs despite their ability to support influenza D virus infection (
74). It is possible that this receptor form is more abundant in the far upper respiratory tract, such as in the nasal cavity. Display in duck tissues of both the respiratory tract and gastrointestinal tract showed that such Sias would represent a Sia form encountered in the avian influenza virus ecological life cycle. The recent identification of a 9-O-acetyltransferase (CAS1 domain-containing protein 1 [CASD1]) will allow a closer examination of regulated expression of the modifying enzyme and the impact on tissue-specific display of 7,9- and 9-O-Ac Sias (
10). In addition, genetic manipulation of expression in cells will allow testing of the functional effects of the modification.
Roles of modified Sia in controlling interactions with infectious pathogens.
Roles of modified Sia in altering the interactions of pathogens with hosts have been known or suggested for many decades—and some strong evidence of effects on influenza virus infection has been reported (
31,
39,
61). Along with the well-known differences in the linkages of the Sias, the modified Sias may further impact influenza viruses at the population level, selecting for HA variants of Sia-binding avidity that may also have additional effects on antigenicity
in vivo (
75).
An additional common modified Sia is
N-glycolylneuraminic acid (Neu5Gc), which is catalyzed from a Neu5Ac-CMP precursor by a CMP-N-acetylneuraminic acid hydroxylase (CMAH) (
76). This modified Sia is present at high levels in many natural influenza virus hosts, including horses and pigs, as well as laboratory hosts such as mice and guinea pigs (
77). However, CMAH has been independently lost by mutation in several influenza virus hosts, including humans and ferrets and likely in Western breeds of dogs (
76,
78,
79). Neu5Gc may to be present but at low levels in tissues of avian lineages (
80–82), but as a CMAH homolog has not been found in birds, this expression may be due to metabolic incorporation from dietary sources. Previous reports have shown that Neu5Gc Sia presented on human cells allows influenza virus binding but may interfere with productive virion entry and infection of some viruses (
83).
In these studies, we examined the distribution of the O-acetylated Sias using the new tools that have become available and did not specifically investigate the presence of the nonmodified forms or of other multiply modified forms, if those were present. That would require either the use of SGRPs that recognize only the nonmodified forms or HPLC analysis of tissue lysates for the presence of modified Sias. Other probes currently under development include CD22-Fc, which shows reduced or blocked Sia binding when the 9-O-Ac modification is present (
84,
85). Previous HPLC analysis has allowed quantification of the ratios of the modified to unmodified Sias. In guinea pig liver tissue, it was seen that Neu5Ac comprises 85% of Sias and that Neu5Gc and Neu4,5Ac
2 modified forms represent 5% and 10% of the remaining Sias, respectively (
24). This may represent an underestimation of the levels due to O-acetyl instability and release during sample manipulation. Quantification by HPLC is an important part of Sia analysis in clarifying the relative levels of abundance but fails to identify and visualize the specific location of display of the modified Sia forms. In our future work, we will seek to explicitly control the levels of the modified Sias by esterase treatments and by modifying enzyme activities so that we can test their role in the infection and release of influenza virus and other viruses.
Summary.
The distribution of several types of modified Sias and their roles in altering the interactions of pathogens with their hosts are still poorly understood decades after the first reports of effects on virus infection and other functions. The use of new tools such as those deployed here and other novel biochemical, genetic, and experimental approaches will allow quantitative analysis of the expression of various Sia moieties and of their effects on pathogens and host responses.