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FACULTY OF MEDICINE AND HEALTH SCIENCES Academic year 2012-2013 THE ROLE OF VIRAL, BACTERIAL, PARASITIC AND HUMAN SIALIDASES IN DISEASE Stefanie VANDE VELDE Promotor: Prof. Dr. Mario Vaneechoutte Dissertation presented in the 2nd Master year in the programme of Master of Medicine in Medicine FACULTY OF MEDICINE AND HEALTH SCIENCES Academic year 2012-2013 THE ROLE OF VIRAL, BACTERIAL, PARASITIC AND HUMAN SIALIDASES IN DISEASE Stefanie VANDE VELDE Promotor: Prof. Dr. Mario Vaneechoutte Dissertation presented in the 2nd Master year in the programme of Master of Medicine in Medicine “The author and the promotor give the permission to use this thesis for consultation and to copy parts of it for personal use. Every other use is subject to the copyright laws, more specifically the source must be extensively specified when using results from this thesis.” 15/04/2013 Vande Velde Stefanie Prof. Mario Vaneechoutte Index 1 Abstract ........................................................................................................................ 1 1.1 Dutch version........................................................................................................ 1 1.2 English version ..................................................................................................... 3 2 Introduction .................................................................................................................. 5 3 Method ......................................................................................................................... 6 4 Results .......................................................................................................................... 6 4.1 Sialic Acids ........................................................................................................... 6 4.1.1 Description and history ..................................................................................... 6 4.1.2 Function ............................................................................................................ 6 4.1.3 Types of sialic acids and distribution ............................................................... 8 4.1.4 The use of sialic acid by different pathogens ................................................... 9 4.2 Sialidases ............................................................................................................ 10 4.2.1 Description ...................................................................................................... 10 4.2.2 The diversity in sialidases and their possible common origin ........................ 10 4.2.3 History ............................................................................................................ 10 4.3 Human sialidases ................................................................................................ 11 4.3.1 Cancer ............................................................................................................. 12 4.3.2 Sialidosis/galactosialidosis ............................................................................. 13 4.3.3 Link between cancer and sialidosis................................................................. 14 4.3.4 Sialidase and synaptic plasticity ..................................................................... 15 4.3.5 Epilepsy .......................................................................................................... 16 4.4 Bacterial and viral sialidases .............................................................................. 17 4.4.1 Influenza ......................................................................................................... 18 4.4.2 Streptococcus pneumoniae.............................................................................. 21 4.4.3 Periodontitis .................................................................................................... 24 4.4.4 Bacterial vaginosis .......................................................................................... 25 4.4.5 Cystic Fibrosis ................................................................................................ 27 4.4.6 Propionibacterium acnes ................................................................................. 28 4.5 5 Trypanosoma cruzi ............................................................................................. 29 Discussion .................................................................................................................. 34 References ......................................................................................................................... 39 1 Abstract 1.1 Dutch version Achtergrond In deze thesis wordt de rol van virale, bacteriële, parasitaire en humane sialidasen in verschillende ziektes besproken. Wat is de exacte rol van deze enzymes en zijn ze een mogelijk target voor nieuwe therapeutische interventies? Sialidasen behoren tot de familie van de exoglycosidasen en katalyseren het verwijderen van siaalzuurresidues van glycoproteïnen en glycolipiden. Humane sialidasen zijn betrokken in het lysosomaal katabolisme en in de modulatie van functionele molecules die gelinkt zijn aan diverse biologische processen (Monti et al. 2002; Miyagi et al. 2004). Verder zijn sialidasen ook teruggevonden in verschillende micro-organismen zoals virussen, bacteriën, protozoa en fungi (Miyagi et al. 2008). Sialidasen hebben een functie in verschillende aandoeningen en om die reden zouden ze een mogelijk target kunnen zijn voor nieuwe behandelingen. Methode In dit literatuuronderzoek werd er gebruik gemaakt van de databases van Pubmed en Web of Science en van de artikels verkregen via de promotor. Resultaten Sialidase activiteit is aantoonbaar in heel wat infectieuze aandoeningen zoals influenza, periodontitis, bacteriële vaginose, luchtweginfecties zoals bij mucoviscidose, en de ziekte van Chagas. De rol van deze sialidasen is erg divers. De enzymes kunnen zowel een rol spelen bij nutritie, invasie, immunosupressie als verspreiding binnen de gastheer. In sommige gevallen hebben ze ook een essentiële rol in biofilmvorming. Daarnaast is sialidase activiteit ook aangetoond in kanker. Neu3, een humaan sialidase, is hierbij significant opgereguleerd. Een defect van Neu1 is aanwezig in de erfelijke metabole aandoeningen sialidose en galactosialidose. Genezing bestaat voorlopig nog niet. Verder zouden stoornissen in de sialidase-activiteit in de hersenen aanwezig zijn bij verschillende psychiatrische en neurologische aandoeningen. 1 Conclusie Voorlopig staat therapie gericht op sialidase-activiteit nog in zijn kinderschoenen. Het probleem hierbij is dat de exacte rol van deze enzymes in veel gevallen nog niet goed gekend is. Binnen eenzelfde organisme kunnen verschillende sialidasen voorkomen, waarvan de functie van de ene al wat duidelijker is dan van de andere. Hoewel op het moleculair niveau, homologieën terug te vinden zijn tussen de sialidasen van zoogdieren, bacteriën, fungi en invertebraten, bestaat er toch een grote biochemische diversiteit (Varki and Schauer 2009). Dit draagt bij tot het feit dat men op zoek moet gaan naar species-specifieke sialidase-inhibitoren in de behandeling van specifieke aandoeningen. Onderzoek is voorlopig enkel nog maar verricht op proefdieren en celculturen, met wisselend resultaat. Meer studies en onderzoek zijn dus een noodzaak. Verschillende studies toonden reeds aan dat humane sialidasen een rol spelen in het ontstaan van kanker. Verder onderzoek naar de pathologische rol van deze sialidasen zou een alternatief kunnen bieden voor de reeds bestaande technieken in de bestrijding van kanker. Sialidose en galactosialidose zijn erfelijke stofwisselingsziekten waar voorlopig nog geen behandeling voorhanden is. 2 1.2 English version Background In this thesis the role of viral, bacterial, parasitic and human sialidases in different diseases will be discussed. What is the exact role of these enzymes and are they a possible target for new therapeutic interventions? Sialidases belong to the family of exoglycosidases and catalyze the removal of sialic acid residues from glycoproteins and glycolipids. Human sialidases are involved in lysosomal catabolism and in the modulation of functional molecules linked to diverse biological processes (Monti et al. 2002; Miyagi et al. 2004). Furthermore, sialidases are also found in various microorganisms including viruses, bacteria, protozoa and fungi (Miyagi et al. 2008). Sialidases display roles in diverse diseases and are therefore a possible target for new treatments. Method This literature research made use of the databases of Pubmed and Web of Science and of the articles obtained by the promoter. Results Sialidase activity is detectable in very different diseases such as influenza, periodontitis, bacterial vaginosis, airway infections such as cystic fibrosis and Chagas disease. The role of these sialidases is very diverse. The enzymes play a role in nutrition, invasion, immunosuppression and spread within the host. In some cases, they display an essential role in biofilm formation. In addition, sialidase activity has been shown to be involved in cancer. Neu3, a human sialidase, is up-regulated significantly in this disease. A Neu1 defect is measurable in the heritable metabolic disorders sialidosis and galactosialidosis, for which there are no cures at present. Furthermore, defects in the sialidase activity of the brain could be involved in different psychiatric and neurological disorders. Conclusion At present, therapy based on sialidase activity is still in its infancy. The problem is that the exact role of these enzymes is mostly not fully understood. Within the same organism, different types of sialidases can be found of which the function is not always that clear. Although at the molecular level, homologies are detectable between enzymes of the mammalian families and those of bacterial, fungal and 3 invertebrate source, a large biochemical diversity in sialidases exists (Varki and Schauer 2009). This means that species-specific sialidase therapy will be needed in the treatment of different disorders. At the present, research on this is only carried out on laboratory animals and cell cultures with varying degrees of success. Furthermore, diverse studies demonstrated the role of human sialidases in the development of cancer. Further investigations on the exact pathological role of these sialidases could provide alternatives for the already existing techniques in the struggle against cancer. Sialidosis and galactosialidosis are heritable metabolic disorders that are not yet curable. 4 2 Introduction Sialic acids are electronegatively charged monosaccharides in higher animals and in a number of viruses, bacteria and eukaryotes. They are involved in many cellular functions and are found at terminal positions of many surface-exposed glycoconjugates, either alone or in oligo- or polymeric form (Varki 1992; Schauer and Kamerling 1997; Schauer 1985). Because of their exposed position, sialic acids are vulnerable to the action of sialidases. Sialidases belong to the family of exoglycosidases and catalyze the removal of sialic acid residues on these surfaceexposed glycoconjugates. Sialidases are not only expressed in humans but also in some viruses, bacteria, fungi and protozoa. In humans, sialidases have a role in lysosomal catabolism but also in the modulation of functional molecules linked to many biological processes (Monti et al. 2002; Miyagi et al. 2004). Disturbance of their expression and biosynthesis can lead to medical problems such as cancer, sialidosis and galactosialidosis. Changes in sialidase activity are also found in neurological and psychiatric disorders such as epilepsy, alcoholism, schizophrenia and severe depression. In pathogens, the role of sialidases is very diverse. The enzymes can be involved in nutrition, invasion, immunosuppression and in the spread of pathogens within the host. In some cases, they play an essential role in biofilm formation. Degradation of human sialic acids by pathogen sialidases is involved in infectious diseases such as periodontitis, cystic fibrosis, pneumonia, vaginosis, and influenza. As a consequence, many efforts have been undertaken to create appropriate pharmacologically active agents. Best known are the competitive inhibitors of sialidases, such as zanamivir and oseltamivir, which hinder budding and spreading of influenza A and B viruses. Inhibitors of bacterial sialidases and trypanosomal transsialidases are urgently needed. The aim of this paper is to provide an overview of the most important diseases in which these sialidases are involved. The role of these enzymes is not always wellunderstood. That is also the reason why this thesis is limited to the diseases for which most information can be found. Furthermore, this work tries to find out whether new treatment options, with sialidase inhibition as a base, can serve as a future solution for different diseases. 5 3 Method The start of this literature study began with a first meeting with Prof. Mario Vaneechoutte. He explained the exact purpose of this paper and introduced the subject by sending some articles. After reading those, I decided to search for more basic information about the subject, using the databases of Pubmed and Web of Science, while using the following key words, alone or in different combinations: ‘sialidase’, ‘neuraminidase’, ‘sialic acid’, ‘biofilm formation’, ‘cystic fibrosis’, ‘Pseudomonas aeruginosa’, influenza’, ‘zanamivir and oseltamivir’, ‘Streptococcus pneumoniae’, ‘Trypanosoma cruzi’, ‘periodontitis’, ‘synaptic plasticity’, ‘cancer’, ‘sialidosis’, ‘galactosialidosis’, ‘bacterial vaginosis’, ‘Propionibacterium acnes’, ‘severe depression’, ‘schizophrenia’, ‘ethanol abuse’ and ‘epilepsy’. Furthermore, relevant articles obtained by references in already found publications and literature suggestions automatically proposed by Pubmed and Web of Science, were used. By reading the abstract, a selection of the most relevant articles was made. 4 Results 4.1 Sialic Acids 4.1.1 Description and history Sialic acids are electronegatively charged monosaccharides in higher animals and in a number of bacteria, viruses and protozoa. They are found at terminal positions of many surface-exposed glycoconjugates, either alone or in oligo-or polymeric form (Varki 1992; Schauer and Kamerling 1997; Schauer 1985). They contribute to the enormous structural diversity of these already complex glycoconjugates, which are major constituents of proteins, lipids of cell membranes and secreted macromolecules (Varki et al. 1999). Figure 1 gives an example of the basic structure of a sialic acid. Figure 1: Structure of a sialic acid (adopted from: http://www.twiv.tv/virus-entry-into-cells) 6 About 75 years ago, sialic acids were discovered by Gunnar Blix, Ernst Klenk and other investigators as a major product released by mild acid hydrolysis of brain glycolipids and salivary mucins. The structure, chemistry and biosynthesis of the compound that they obtained were revealed in the 1950s and 1960s by multiple groups. Partly because of its discovery in salivary mucins (Greek: sialos), this family was named the ‘sialic acids’ (Varki and Schauer 2009). 4.1.2 Function Sialic acids play important roles in a wide range of biological processes, including cell-cell and small molecule-cell recognition (Severi et al. 2007). It is therefore believed that the appearance of these monosaccharides has facilitated the evolution of higher organisms. There is almost no biological event in mammals in which these compounds are not involved. Therefore, errors in their biosynthesis or degradation have dramatic biological consequences and may lead to diseases including cancer (Varki 1992). It should be mentioned here already that Neu5Gc, an important sialic acid in ‘great apes’ and other mammals, is absent in humans because of an inactivating mutation in the cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene (CMAH). As a result, humans seem to be resistant to infectious diseases, in which a pathogen or bacterial toxin specifically binds to Neu5Gc (Varki 2009). Due to the surface location of the acidic molecules, sialic acids shield macromolecules and cells from enzymatic and immunological attacks (Schauer 2004). It is therefore perhaps not surprising that many pathogenic bacteria also decorate their cell surfaces with sialic acids. This results in important phenotypes regarding their ability to resist the host’s innate immune response and their ability to interact particularly with different host-cell surfaces. As a consequence, sialic acids empower pathogens to enter the host cell (Severi et al. 2007). For example, capsuledeficient or nontypeable (NT) Haemophilus influenzae is known to be the most common cause of otitis media in young children. Bouchet et al. (2003) reported that NT H. influenzae isolates have the potential to incorporate sialic acid from the host into their lipopolysaccharide (LPS). Strains expressing sialic acid are more resistant to the bactericidal activity of normal human serum in vitro (Hood et al. 1999). 7 4.1.3 Types of sialic acids and distribution Sialic acids are a family of monosaccharides, containing about 50 members, which are derived of neuraminic acid. N-Acetylneuraminic acid (Neu5Ac) and Nglycolylneuraminic acid (Neu5Gc) are the two most frequently occurring members of the sialic acid family in mammals, with the notable exception of our species (see above). Figure 2 shows their structure. Figure 2: Structure of Neu5Ac and Neu5Gc (adopted from: http://www.nature.com/nature/journal/v446/n7139/fig_tab/nature05816_F2.html). The most abundant and best-studied sialic acid is Neu5Ac (Angata and Varki, 2002). Never have all kinds of sialic acid been found in one cell or organism. The distribution depends on the animal and cell species as well as on the function of a cell and seems to be strongly regulated on the gene level. The animal with the highest amount of different sialic acids known so far is the cow (Schauer and Kamerling 1997). In man, the number of sialic acid types is much smaller, with Neu5Ac as the most dominant one, followed by derivatives which are O-acetylated and O-lactylated at the sialic acid side chain (Schauer and Kamerling 1997). As mentioned earlier, Neu5Gc is absent in humans because of an inactivating mutation of the CMAH gene. As a consequence, humans seem to be resistant to infectious diseases, in which a pathogen or bacterial toxin specifically binds to Neu5Gc (Varki 2009). The human malarial parasite Plasmodium falciparum preferentially 8 recognizes Neu5Ac in the process of red blood cell invasion (Martin et al. 2005). In contrast, the corresponding major binding protein of the chimpanzee/gorilla malarial parasite Plasmodium reichenowii preferentially recognizes Neu5Gc. This may explain why humans and chimpanzees are relatively or absolutely resistant to the malarial pathogen derived from each other (Blacklock and Adler 1922; Rodhain 1939). Additionally, Varki and coworkers hypothesized that the long-term dietary intake of Neu5Gc (red meat and milk products) with incorporation into human tissues could combine with the circulating anti-Neu5Gc antibodies, to stimulate chronic inflammation (Varki 2007). 4.1.4 The use of sialic acid by different pathogens Bacteria have two primary routes to obtain sialic acid. The first route of acquiring sialic acid is de novo biosynthesis, as used by a number of bacteria including Escherichia coli K1, Campylobacter jejuni and Neisseria meningitidis (Vimr and Lichtensteiger 2002). The second source of sialic acid is the environment (Vimr et al. 2004). In this paper, we consider the mammalian host as the environment. Many pathogens secrete a sialidase that releases sialic acid from a diverse range of host sialoglycoconjugates (Corfield 1992). In contrast, other sialic acid-utilizing bacteria, such as the respiratory pathogen H. influenzae, lack genes for a sialidase although they are dependent on host derived sialic acid (Bouchet et al. 2003). NT H. influenzae expresses at least 3 sialyltransferases. These enzymes transfer sialic acid from the host glycoproteins to the surface (LPS) of the pathogen (Hood et al. 2001, Vimr et al. 2000). Most likely, these pathogens use free sialic acid that is made available by other sialidase-expressing bacteria living in the same niche (Shakhnovich et al. 2002). Another hypothesis by Sohanpal et al. (2007) puts that host sialidases, that are activated in the course of inflammation, are used. In summary, whether sialic acid is synthesized de novo or obtained exogenously, many pathogens are able to decorate their surface molecules (LPS and capsular polysaccharides) with sialic acid to mimic host cell surfaces. This ‘moleclar mimicry’ helps in the avoidance of host immune attack (Vimr and Lichtensteiger 2002; Severi et al. 2007). 9 4.2 Sialidases 4.2.1 Description Sialidases catalyze the removal of sialic acid residues from glycoproteins and glycolipids, which is the initial step in the degradation of these glycoconjugates. Sialidases exist in general in metazoan animals, from echinoderms to mammals, and are also found in various viruses and other microorganisms including bacteria, fungi and protozoa and even in forms mostly lacking sialic acids (for example Streptococcus pneumoniae) (Miyagi et al. 2008). Sialidases of mammalian origin have been involved not only in modulation of functional molecules linked to many biological processes but also in lysosomal catabolism since sialidase deficient individuals develop lysosomal storage diseases such as sialidosis and galactosialidosis (Monti et al. 2002; Miyagi et al. 2004). Otherwise, in microorganisms the same enzymes appear to play roles related to nutrition and virulence (Corfield 1992). 4.2.2 The diversity in sialidases and their possible common origin The proposal of a common sialidase origin in higher animals is suggested by the presence of apparently homologous enzymes in this kingdom. Since homologies at the molecular level are also detectable between enzymes of the mammalian families and those of bacterial, fungal and invertebrate sources (Varki and Schauer 2009), this props up the idea that some pathogens may have acquired the genetic information during association with their animal hosts. Horizontal gene transfer between animals and pathogens seems possible. Some finding indicate that sialidase genes were recently transferred via phages among bacteria (Varki and Schauer 2009; Roggentin et al. 1993). Most mammalian and bacterial sialidases share a set of common ‘Asp boxes’ (Ser-XAsp-X-Gly-X-Thr-Tyr) that, together with a number of other highly conserved amino acids, are probably involved in the maintenance of the enzyme protein conformation (Varki and Schauer 2009). 4.2.3 History In the 1940s, sialic acid had been shown to be the cellular receptor for influenza. As Clostridium perfringens and Vibrio cholera were able to destroy the receptor sites for 10 the influenza virus on the surface of the human red cell, the responsible enzyme was named the ‘receptor-destroying enzyme’. It was later shown that the ‘receptordestroying enzyme’ acts as a sialidase (Varki and Schauer 2009). Another group found a similar activity in other bacteria. Alfred Gottschalk suggested the name ‘neuraminidase’ for this activity in 1957 (Varki and Schauer 2009). Sialidase activity in higher organisms was detected for the first time in commercial preparations of bovine and human glycoproteins (Warren and Spearing 1960). From that time on, several reports have demonstrated its presence in a wide variety of mammalian cells and tissues. Carubelli et al. (1962) detected sialidase activity in soluble fractions isolated from different tissues of rats, and several papers described its presentation in lysosomes (Mahadevan et al. 1967). Next, it was detected in plasma membranes (Schengrund and Rosenberg 1970) and Golgi fractions (Kishore et al. 1975). However, in the early days, it remained uncertain whether the activities originated from the same or different types of sialidase. This was partly because of molecular instability and low levels of expression (Miyagi et al. 2012). 4.3 Human sialidases At least four sialidase homologs have been identified in the human genome, namely NEU1, NEU2, NEU3 and NEU4. All enzymes have in common several Asp-boxes and the RIP/RPL motif (Arg-Ile/Leu-Pro) that is also discovered in microorganisms (Miyagi et al. 2008; Achyuthan et al. 2001). The four sialidases possess different substrate specificities. In particular, NEU1 hardly hydrolyzes gangliosides and NEU3 acts preferentially on gangliosides but not on glycoproteins. It is a characteristic feature of NEU4 that it operates on mucin (Seyrantepe et al. 2004; Yamaguchi et al. 2005). Although many functional aspects are not fully understood, recent progress in gene cloning has facilitated clarification of important biological roles in cellular functions including cell differentiation, cell growth and apoptosis (Miyagi et al. 2012). The four sialidases differ in their subcellular localization and enzymatic properties Neu1, Neu2 and Neu3 are known to be localized predominantly in the lysosomes, cytosol and plasma membranes, respectively. Neu4 is found in lysosomes or in mitochondria and the endoplastic reticulum. NEU1 generally shows the strongest expression, 10–20 times greater than that of NEU3 and NEU4, whereas NEU2 11 expression is extremely low at the most at only one 4000th to 10,000th of the NEU1 value in a range of tissues (Hata et al. 2008). 4.3.1 Cancer Observations on sialidase activity in cancer cells have suggested that endogenous sialidase might be related to transformation and tumour invasiveness. In fact, the four types of mammalian sialidases have been found to behave in different manners during carcinogenesis (Miyagi et al. 2004). Three of the sialidases, Neu1, Neu2 and Neu4 show a tendency of down-regulation, while Neu3 showes marked up-regulation (Yamanami et al. 2006). NEU1 Kato et al. (2001) introduced NEU1 into B16 melanoma cells, resulting in suppression of experimental pulmonary metastasis and tumor progression. This caused a reduction in anchorage-independent growth and increased sensitivity to apoptosis. Likewise, overexpression of the human NEU1 in the colon adenocarcinoma HT-29 case, resulted in a suppressed cell migration and invasion, whereas its knockdown resulted in the opposite. When NEU1-overexpressing cells were injected trans-splenically into mice, the in vivo liver metastatic potential was reduced significantly (Uemura et al. 2009). NEU3 The human orthologue NEU3 is markedly up-regulated in various cancers. NEU3 is known to suppress apoptosis in cancer cells (Miyagi et al. 2008). In addition, its overexpression causes impaired glucose tolerance and hyper-insulinaemia together with overproduction of insulin in enlarged islets in transgenic mice (Sasaki et al. 2003). Recent epidemiological reports (Nishii et al. 2001; Yoshida et al. 2006) describing higher incidence of cancers in diabetic patients than in controls, have suggested that these diseases might be closely related to each other in pathogenesis. In this context, it is feasible that NEU3 possibly regulates common signaling pathways involved in pathogenesis of the both diseases. Further clarification of the pathological roles of NEU3 should lead to potential applications in control of cancer and diabetes (Miyagi et al. 2008). 12 4.3.2 Sialidosis/galactosialidosis Neu1 is only catalytically active as a component of a high molecular weight, multiprotein complex, containing PPCA (carboxypeptidase protective protein/cathepsin A), ß-galactosidase and N-acetylgalactosamine-6-sulfate sulfatase, in lysosomes (Verheijen et al. 1982; Yamamoto and Nishimura 1987; Pshezhetsky and Poitier 1996). Dissociation leads to sialidase inactivation (D’Azzo et al. 1982). NEU1 is linked to two neurodegenerative lysosomal storage disorders: sialidosis and galactosialidosis. Both are autosomal recessive diseases. Residual neuraminidase activity in patients with sialidosis or galactosialidosis is typically < 1 % of normal levels (d’Azzo et al. 1995). Sialidosis is caused by defects in the genomic DNA, including frameshift insertions and missense mutations. Galactosialidosis is marked by a combined deficiency of Neu1 and β-galactosidase due to the absence of a functional PPCA, leading to deficient enzyme activity (Bonten et al. 199; Pshezhetsky et al. 1997). In both cases, the catabolic pathway for degradation of sialylated glycoconjugates is disrupted, causing their accumulation in the lysosome and excretion in urine (Thomas 2001; D’Azzo et al. 2001). The different clinical phenotypes Diverse clinical phenotypes exist, varying in the onset and severity of the symptoms. Type I sialidosis, which is also called the cherry-red spot/myoclonus syndrome, is a relatively mild disease that occurs in the second decade of life. This type of sialidosis results in progressive loss of vision associated with nystagmus, ataxia and grand malseizures but not dysmorphic features (Thomas and Beaudet 1995). Type II sialidosis is the severe form of the disease characterized by the presence of abnormal somatic features, including coarse facies and dyostosis multiplex. Type II sialidosis is divided into three subtypes: (i) congenital or hydropic (in utero); (ii) infantile (0-12 months) and (iii) juvenile (2-20 years) (Thomas and Beaudet 1995). The congenital form is associated with either hydrops fetalis and death in utero or neonatal ascites and death at an early age. Features include facial edema, inguinal hernias, hepatosplenomegaly, stippling of the epiphyses and periosteal cloaking. As for sialidosis patients, galactosialidosis patients are diagnosed with either an early infantile, late infantile or a juvenile/adult form of the disease, based on age at onset and severity of clinical manifestations. The early infantile form of galactosialidosis is clinically very similar to the congential type II form of sialidosis. Both are characterized by visceromegaly, hydrops fetalis, ascites and early death. The late infantile/childhood forms of 13 galactosialidosis and sialidosis are also similar, with the exception of milder neurological involvement in the galactosialidosis patient (d’Azzo et al. 1995). Neu4 and gene replacement therapy? Seyrantepe et al. (2004) illustrated that the human sialidase Neu4, displays broad substrate specificity and trafficking to the lysosomal lumen. Overexpression of NEU4 cleared storage materials from cultured fibroblasts of sialidosis and galactosialidosis patients. Their data also showed that Neu4 is active against a majority of endogenous substrates of Neu1. Being expressed in Neu1 deficient sialidosis fibroblasts, Neu4 completely eliminated undigested substrates of Neu1 and restored normal morphological phenotype of the lysosomal compartment, therefore offering therapeutic potential (Seyrantepe et al. 2004). Seyrantepe et al. (2004) observed that complete elimination of storage materials happened in 55% of sialidosis cells and in 25% of galactosialidosis cells while only 3–5% of cells were transfected with NEU4 plasmid. These data show that the Neu4 released from the transfected cells, enters cells neighboring the Neu4-expressing cells and corrects their phenotype. Therefore, recombinant human Neu4 might be of potential use for enzyme replacement therapy in sialidosis and galactosialidosis. Still, enzyme replacement rarely achieves superphysiologic levels of the enzyme in target tissues, and because patients with Neu1 deficiency but with two normal Neu4 alleles still develop the disease, physiologic levels of Neu4 are likely not sufficient to prevent accumulation of sialylated compounds. Much more attractive, therefore, would be to induce the expression of the endogenous NEU4 gene to compensate for NEU1 deficiency. Northern blotting revealed Neu4 expression in every human tissue examined suggesting that such an approach may have a general effect throughout the whole organism (Seyrantepe et al. 2004). 4.3.3 Link between cancer and sialidosis The first clinical observation that suggested a possible link between a decreased activity of lysosomal sialidase and the development of a variety of neoplasms, came from Yagi and coworkers (2011). In a family where three of four siblings expressed mutants of NEU1 responsible for decreased sialidase activity, there was not only a link to sialidosis type I phenotype, but also to the occurrence of neoplasms of different origins. Except for patients 1–3, no other family members had sialidosis, and except for patients 1–3, no other family members had any kind of malignancy. 14 Environmental conditions that may have facilitated the neoplasm development in their birthplace were not found either (Yagi et al. 2011). An earlier report (Uchihara et al. 2010) included the finding obtained from an autopsied patient with sialidosis type I, who died due to intractable lymphoma at the age of 32. This comorbidity was initially considered coincidental. Subsequent development of other kinds of neoplasms in the siblings however, could not be attributed to only coincidence, considering the extreme rarity of sialidosis (Yagi et al. 2011). The occurrence of neoplasms in these cases raised the possibility that either biallelic mutations in the relevant gene or the resultant decrease in the sialidase activity is linked to the development of these neoplasms (Yagi et al. 2011). Indeed, it has been reported that a decreased expression of the Neu1 protein is associated with an enhanced metastatic ability of mouse colon adenocarcinoma cells (Sawada et al. 2002). It may also be considered that decreased sialidase activity, which is also detected in galactosialidosis, may also be associated with the development of neoplasm. So far, no report has been published to verify this hypothesis, suggesting that some consequences of NEU1 mutations are mediated by mechanisms other than decreased sialidase activity. Yagi et al. (2011) estimate that the relationship between sialidosis and neoplasms may be the result of the diminished activity of sialidase, but the fact that no galactosialidosis cases with neoplasm have been reported suggests other possible mechanisms. More such reports are necessary to clarify the details of this relationship. 4.3.4 Sialidase and synaptic plasticity Cell adhesion molecules are essential in neuronal network formation during development and adult synaptic plasticity (Venero et al. 2006). In the brain, neural cell adhesion molecules (NCAMs) are primarily expressed by neurons. Polysialic acid (PSA), a sialic acid polymer, is associated with these NCAMs. Modulation of the level of PSA influences synaptic plasticity, neurite growth and cell migration (Seki and Arai, 1993). PSA levels are high during embryonic development, whereas PSA expression in the adult is generally restricted to the hippocampus (Takahashi et al. 2012). The enzyme involved in the degradation of PSA remained uncertain for a 15 long time until Takahashi and coworkers (2012) identified NEU4 as the possible regulator of PSA levels. Changes in the state of sialylation of NCAMs were demonstrated in inflammatory diseases and as a result of exposure to neurodegenerative factors such as ethanol and drugs (Poluektova et al. 2005; Mackowiak et al. 2007; Azuine et al. 2006). Azuine et al. (2006) demonstrated that increased sialidase-dependent degradation of brain gangliosides may be responsible for psychological and neurological impairment in the brain caused by ethanol. Contrary to ethanol exposure, chronic stress increases PSA NCAM and decreases sialidase-activity, and causes both hippocampal atrophy and impairment of learning. It may explain the general opinion that sialidase is involved in psychiatric disorders like severe depression and schizophrenia (Kandel 2001; Wielgat et al. 2011; Gilabert-Juan et al. 2012). Intensive polysialylation of NCAM was also noted in hippocampal areas of Alzheimer’s disease and in the epileptic temporal lobe (Boyzo et al. 2003; Mikkonen et al. 1999). 4.3.5 Epilepsy Epilepsy is a serious neurological disorder characterized by recurrent, unprovoked seizures. Almost 1% of humans suffer from epilepsy during their lifetime, usually children and people over the age of 65 years (Holmes 1997). While in many cases epilepsy is a mild condition with a favorable outcome, epilepsy can also be life threatening if the seizure is prolonged (status epilepticus) (Logroscino et al. 2008). Many causes and types of epilepsy exist, which is also the reason why treatment can be quite difficult (Holmes 1997). The past few decades, more than 20 new antiepileptic drugs have been accepted (Lasoñ et al. 2011). Many of the antiepileptic drugs that are presently available, have adverse side effects such as alterations in cognition and behavior. Consequently, their use is limited (Jokeit and Ebner 2002). In addition, nearly one third of patients are refractory to antiepileptic drugs and have continued seizures despite appropriate dosing (Kwan and Brodie 2000). Thus, searching for novel therapeutic strategies remains a priority in drug development. One of the promising new ways to regulate abnormal neuronal excitability is to alter sodium channel activation through the modification of the negative surface charge of the cellular membrane from polysialic acid. It has been shown that negatively 16 charged sialic acid residues located close to pores of voltage-gated sodium channels substantially influence their gating properties (Messner et al. 1985). Isaev and coworkers (2007) showed that desialylation of hippocampal slices with neuraminidase distorted the action potential threshold, delayed the onset of epileptiform activity and reduced the population spike frequency in the CA3 zone of rat hippocampus. These findings suggest that modulating surface charges by targeting negatively charged sialic acids may be an effective strategy to treat status epilepticus (Isaeva et al. 2011). 4.4 Bacterial and viral sialidases Bacterial sialidases are involved in the bacterial invasion into the host and the spread within the host. The enzyme activity belongs to the initial step in the degradation of sialic acids. Bacterial neuraminidases are a superfamiliy of multi-domain enzymes and are often secreted as soluble proteins or are bound to the bacterial surface. Sometimes, they are not secreted at all. The sequential accordance in bacterial neuraminidases is around 30 % and is therefore very low (Schwerdtfeger and Melzig 2010). Nevertheless there are two conserved motives: the RIP/RPL-motive (ArgIle/Leu-Pro) and the Asp-Box-motive (Ser-X-Asp-X-Gly-X-Thr-Tyr). The arginine of the RIP/RPL motive interacts with the substrate’s carboxyl group and the Asp-box might be involved in the secretion process of the bacterial neuraminidases (Taylor 1996). The proteins differ in form and in size between the species. The size ranges from 40 kDa up to 120 kDa. Usually the enzyme is a monomer, but there are descriptions of oligomeric structures (Schwerdtfeger and Melzig 2010). Like the bacterial neuraminidases, the influenza neuraminidase has a catalytic centre that is highly conserved in the different subtypes. The sequence homology between the influenza and the bacterial neuraminidase is 15% (Schwerdtfeger and Melzig 2010). In contrast with the bacterial enzymes, viral neuraminidases have no Asp box. This results in small differences in the active site resulting in differing kinetics, binding affinity and substrate preference. These differences make pathogen-specific neuraminidase inhibitors possible (Taylor 1996). 17 4.4.1 Influenza Influenza is a globally important viral infection. About 20% of children and 5% of adults worldwide develop symptomatic influenza A or B each year (Turner et al. 2003). Influenza goes from symptomless infection through various respiratory syndromes, disorders affecting the lung, heart, brain, liver, kidneys, and muscles, to fulminant primary viral and secondary bacterial pneumonia. Most influenza infections are spread by virus loaded respiratory droplets that are expelled during coughing and sneezing (Nicholson et al. 2003). Influenza viruses have segmented genomes and show great antigenic diversity. Of the three types of influenza viruses (A, B, and C), only types A and B cause widespread outbreaks. Influenza A viruses are classified into subtypes based on antigenic differences between their two surface glycoproteins, haemagglutinin (H) and neuraminidase (N). A total of 15 haemagglutinin subtypes (H1–H15) and nine neuraminidase subtypes (N1–N9) have been identified for influenza A viruses. Only one subtype of haemagglutinin and one of neuraminidase are recognized for influenza B viruses. Figure 3 shows the general structure of the influenza virus. 18 Figure 3: Structure of the influenza virus (adopted from: http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html) Haemagglutinin facilitates entry of the virus into host cells through its attachment to sialic-acid receptors. An important function of neuraminidase is to release newly synthesized virus by cleaving sialic acid from host cell glycoproteins during budding. This sialidase activity also serves to prevent aggregation of virions to each other and to the mucins in the respiratory tract (Nicholson et al. 2003). The epidemiological behavior of influenza in people is related to the two types of antigenic variation of its envelope glycoproteins: antigenic drift and antigenic shift. During antigenic drift, new strains of virus develop by accumulation of point mutations in the surface glycoproteins. This feature enables the virus to escape immune recognition, leading to repeated outbreaks during interpandemic years. Antigenic shift occurs with the appearance of a new and potentially pandemic influenza A virus that possesses a novel haemagglutinin alone or with a novel neuraminidase. The new virus is antigenically distinct from earlier human viruses and could not have arisen from them by mutation (Nicholson et al. 2003). Figure 4 and 5 show a more clear picture of the terms ‘antigenic drift’ and ‘antigenic shift’ 19 Figure 4: Antigenic drift (adopted from: http://homepage.usask.ca/~vim458/virology/studpages2009/H1N1/Drift.html) Figure 5: Antigenic shift (adopted from: http://homepage.usask.ca/~vim458/virology/studpages2009/H1N1/Drift.html 20 Influenza and bacterial complications It has been known for more than a century that respiratory viruses predispose to bacterial complications. This association came into particular focus as the influenza pandemic of 1918 took probably more than 20 million of lives, more than World War I (Nicholson et al. 1998). Although the primary pneumonitis seen during this pandemic was severe, the majority of patients died of secondary bacterial pneumonia (Taubenberger et al. 2001), and bacteria were frequently detected in the postmortem examination of the lungs (Muir & Wilson 1919; Nicholson et al. 1998). This outbreak was the beginning of investigations into the epidemiology and pathology of bacterial-viral interactions that continue today. DAS181, a novel candidate therapeutic agent against the influenza virus DAS181 is a sialidase fusion protein in clinical development as a broadspectrum therapeutic and prophylactic treatment against the influenza virus and the parainfluenza virus (Hedlund et al. 2010). By cleaving sialic acids from the host cell surface, DAS181 inactivates the host cell receptors recognized by both viruses (AhTye et al. 1999), making the host cells more resistant to influenza and parainfluenza infection (Jedrzejas 2001). Furthermore, DAS181 reduces bacterial colonization, proving that desialylation per se does not increase susceptibility to secondary bacterial infection. Hedlund and coworkers (2010) belief that subsequent bacterial infection after influenza infection is not so much due to the desialylation by the influenza virus but by the increased epithelial denudation as a result of viral infection (Hedlund et al. 2010). 4.4.2 Streptococcus pneumoniae Streptococcus pneumoniae (the pneumococcus) is the most common agent causing community-acquired pneumonia, otitis media in children and sepsis and meningitis in adults after influenza infection. Subsequent bacterial infections may be the result of the weakened functions of immunologic cells after viral infection. Leukopenia is a common finding during influenza in humans. Influenza virus in combination with S. pneumoniae causes more apoptosis of neutrophils than either pathogen alone (Engelich et al. 2001). Like the influenza virus, the pneumococcus possesses sialidase activity (Simonsen 2001). Many cellular structures that can act as bacterial receptors are covered by sialic acids on cell surface carbohydrates. If sialic acids are cleaved by a bacterial or 21 viral neuraminidase, bacteria may be able to adhere and invade (Okamoto et al. 2003). The S. pneumoniae genome encodes up to three neuraminidases named NanA, NanB and NanC. NanA and Nan B may serve distinct functions as their localization and cleavage specificity differ. Since recently, NanA is suggested to be a hydrolytic enzyme with broad specificity for different sialic acid linkages (Xu et al. 2008). NanB, on the other hand, is secreted and has strict specificity for α 2-3-linked sialic acid, suggesting that the enzyme is an intramolecular trans-sialidase (Gut et al. 2008). However, the exact role of NanB remains vague (King 2010). A study of clinical isolates of S. pneumoniae showed NanA, NanB, and NanC to be present in 100%, 96%, and 51% of strains, respectively, with NanC more prevalent in cerebrospinal fluid (Pettigrew et al. 2006). Some isolates from invasive disease lack both NanB and NanC, bringing into question the requirement for either of these loci (King 2010). Biofilm formation and sialidase activity in S. pneumoniae A biofilm consists of a structured population of microorganisms adhered to a surface and embedded in an extracellular matrix consisting mainly of exopolysaccharides (Hall-Stoodley and Stoodley 2009). Biofilms show a modified phenotype in terms of their growth rate and gene expression patterns. Microorganisms growing in biofilms can be up to 1000 times more resistant to antibiotics than the same free-living microorganisms. In addition, they are also very resistant to phagocytosis, making biofilms extremely difficult to eradicate from living hosts (Lewis 2001). As in many other bacterial infections, S. pneumoniae is known to grow in biofilms (Moscoso et al. 2009). It is likely a multifactorial process, possibly including DNA, pneumococcal proteins and capsular polysaccharide. Furthermore, there is increasing evidence that modification and utilization of host sugars contribute to biofilm formation (King 2010). NanA and NanB expression is increased in pneumococcal biofilms (Trappetti et al. 2009). In addition, NanA and NanB mutants have a reduced ability to form a biofilm in vitro. No role was identified for NanC (Parker et al. 2009). Recent work proposes that free sialic acid contributes to efficient pneumococcal biofilm formation. Addition of sialic acid to an in vitro biofilm model significantly increased the number of adherent bacteria, whereas the addition of 26 other sugars had no effect (Trappetti et al. 2009). These data suggest that sialic acid may serve as a signaling molecule that increases the capacity of S. pneumoniae to 22 form a biofilm or sialic acid could be a component of the biofilm matrix (King 2010). The effect of oseltamivir and zanamivir treatment on pneumococcal infection Early oseltamivir treatment (selective influenza neuraminidase inhibitor) reduced the development of acute otitis media by 44% in influenza infected children 1 to 12 years old (Whitley et al. 2001). In another study (Treanor et al. 2000) early oseltamivir treatment of influenza in healthy adults ages 18 to 65 years, reduced occurrence of secondary complications (otitis media, sinusitis, bronchitis or pneumonia) as well as antibiotic use by 50%. One study (MIST study group 1998) included a group of patients with an elevated risk for complications from influenza caused by factors like mild asthma or age above 65 years. In this group zanamivir treatment (selective neuraminidase inhibitor) of influenza reduced the incidence of complications from 46% to 14% and antibiotic use from 38% to 14%. A wealth of information is available about how the inhibitors zanamivir and oseltamivir carboxylate (OC) act on the influenza NA. In contrast, there are no data that describe how they inhibit bacterial NA that can give an explanation for the beneficial effects of treatment with these drugs in mouse models of pneumococcal infection. Gut et al. (2011) described the crystal structures of pneumococcal NanA (involved in biofilm formation and colonization) in complex with zanamivir and OC and compared it with the binding modes of the inhibitors in the viral enzyme. NanA complexes with zanamivir and OC show that although distinct from the influenza virus NA active site, the NanA active site has high enough plasticity to accommodate the influenza-virus specific inhibitors. Even though inhibitors have only weak (zanamivir) and medium (OC) inhibitory effects, it seems that a slight reduction in NanA activity can have a dramatic effect on S. pneumoniae colonization (Gut et al. 2011). In some pediactric cases in Japan, mortalities and neuropsychiatric events have been reported with the use of oseltamivir. They suggested that these drugs also inhibit endogenous enzymes involved in sialic acid metabolism in addition to their inhibitory effects on the viral sialidase (Fuyuno 2007, Maxwell 2007). Hata et al. (2008) examined whether these inhibitors might indeed affect the activities of human sialidases. Human sialidases differ in primary structures and enzyme properties but possess tertiary structurs similar to those of the viral enzymes. Using recombinant 23 enzymes corresponding to the four human sialidases identified so far, they found that OC scarcely affected the activities of any of the sialidases. 4.4.3 Periodontitis Periodontitis is a major cause of tooth loss. It is known as a prevalent chronic disease that affects up to 80% of the adult population worldwide (Li et al. 2012). Two of the pathogens involved in this infection are Tannerella forsythia and Porphyromonas gingivalis (Socransky et al. 1998). T. forsythia sialidase and biofilm formation NanH sialidase is the major sialidase expressed by the bacterium T. forsythia. This bacterial sialidase is essential for initial attachment to the glycoproteins present in host cells or in the oral environment (Roy et al. 2011). Honma and coworkers (2011) showed that the T. forsythia sialidase is important in interactions with human gingival epithelial cells in a mechanism that may expose cryptitopes by unmasking sialic acid-masked epitopes for adhesion and invasion. In addition, a recent study has demonstrated that sialic acid released from the sialylated glycoconjugates by NanH sialidase action may serve as a growth factor for biofilm growth (Roy et al. 2010). T. forsythia sialidase can be inhibited by oseltamivir (Roy et al. 2010). Roy and coworkers (2011) tested the effect of this inhibitor on biofilm formation to examine whether any or all of the growth was dependent on sialic acid. They demonstrated that biofilm growth and initial adhesion with sialylated mucin and fetuin were inhibited two-to threefold by the sialidase inhibitor oseltamivir. A similar reduction (three – to fourfold) was observed with a NanH mutant compared with the wild-type. These data highlight the roles of sialic acid as a source of nutrition and as a receptor in the initial stage of biofilm formation on glycoprotein-coated surfaces, a situation which may mimic in vivo conditions (Derrien et al. 2010). P. gingivalis sialidase and biofilm formation Like T. forsythia, P. gingivalis also exhibits neuraminidase activity (Moncla et al. 1990). However, little is known about the enzyme responsible for this activity. Recently, Li and coworkers (2012) identified a gene (PG0342) encoding a neuraminidase in P. gingivalis. They revealed that the PG0352 deletion mutant failed to produce an intact capsule layer and that the mutant formed less biofilm and was less resistant to killing by the host complement. 24 A possible role for sialidase in periodontal pathogen interactions In addition to interactions with host cells in the oral cavity, periodontal pathogens also show various interactions with other oral bacteria in the formation of biofilms. Recent evidence suggests that sialic acid and sialidases are involved here as well (Stafford et al. 2012). Pretreatment of P. gingivalis with sialidase reduces interactions with Streptococcus sanguinis (Stinson et al. 1991). New proof proposed that a nanH mutant of T. forsythia aggregates less well to the sialic-acid coated bridging organism Fusobacterium polymorphum (Bolstad et al. 1996), suggesting a nutritional and physical basis for their synergistic relationship (Sharma et al. 2005). 4.4.4 Bacterial vaginosis Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a complex bacterial milieu; it is the most common disorder diagnosed in women who are examined at sexually transmitted disease clinics and is reported in about 20% of pregnant women (Cauci et al. 1998). BV is characterized by a shift in vaginal microflora, with a decrease in the prevalence of Lactobacillus (especially those that produce hydrogen peroxide) and an increase in the prevalence and concentration of anaerobic bacteria like Gardnerellla vaginalis and Mycoplasma hominis (Cauci et al. 2003). These anaerobic bacteria produce enzymes and decarboxylases that degrade proteins and convert the amino acids into amines. These amines raise the vaginal pH and produce a characteristic fishy odor. The diagnosis of bacterial vaginosis is based on the Amsel criteria: a vaginal pH more than 4.5, a characteristic milky discharge, a positive ‘whiff test’ (amines on the vaginal discharge produce a fishy odor with 10% KOH) and the presence of ‘clue cells’ (bacteria-coated vaginal epithelial cells) (Swidsinski et al. 2005). BV in pregnancy is associated with several adverse outcomes including spontaneous abortion, preterm delivery (< 37 weeks’ gestation), early preterm delivery (< 32 weeks’ gestation), premature rupture of membranes, low birth weight (< 2500 g at birth), amniotic fluid infections, chorioamnionitis, and postpartum and post-surgery infections. In addition, BV has been associated with upper genital tract infections, urinary infections, and an increased risk of sexually transmitted diseases, including human immunodeficiency virus (HIV) and herpes simples virus-2 infection (Cauci et al 2008). 25 Absence of inflammation in BV A characteristic of BV is the scarcity of leukocytes and inflammatory signs. Although BV is not an inflammatory condition, increased vaginal concentrations of IL-1ß have consistently been found in women with BV (Cauci et al. 2002; Cauci et al 2003). IL-1ß is a well-known proinflammatory master cytokine, activated in the early response to infection, which is able to induce several other cytokines and recruit different types of white cells. Therefore, it appears paradoxical that women with BV do not show significant accumulation of vaginal white cells, especially neutrophils, which are the main leukocytes in vaginal secretions. The absence of massive neutrophil accumulation, which is the most striking characteristic of BV in contrast to other vaginal infections, could permit bacteria to ascend to the upper genital tract (Cauci et al. 2008). Sialidases and their role in BV Some bacteria involved in BV are able to produce sialidases. Since both cervical mucus and amniotic fluid have been demonstrated to contain significant amounts of sialic acid, sialidases may promote virulence by enhancing the ability of these bacteria to adhere to, invade, and destroy mucosal tissue (Briselden et al. 1992). Furthermore, sialidase and prolidase, both produced by BV-associated bacteria, are shown to play a role in the down-regulation of the vaginal adaptive immunity (Cauci et al. 1998). Sialidases and prolidases are potentially capable to degrade several key mucosal protective factors, such as mucins, cytokines, immunoglobulins, antimicrobial molecules, and host cell receptors. Sialic acid commonly occupies the terminal position of carbohydrate moieties attached to several mucosal defense factors such as secretory IgA, secretory component, lactoferrin, secretory leukocyte protease inhibitor, and others (Perrier et al. 2006). Sialylation appears crucial for the recognition of microbial molecular patterns by local host defense proteins. Potentially, the combined action of different hydrolytic enzymes such as sialidases and prolidases can deregulate several crucial host antimicrobial/ immune responses, creating a local immunosuppression. In other words, sialidases and/or prolidases directly or indirectly could cause an inefficient immune cascade after an IL-1ß rise, ending in low neutrophils counts (Cauci et al. 2008). 26 Biofilm formation in BV Swidsinki and coworkers (2005) found that BV is associated with the development of a biofilm containing an abundance of Gardnerella vaginalis bacteria. In contrast to BV, adherent biofilms containing great quantities of G. vaginalis were not observed on the epithelium of most healthy women (Swidsinki et al. 2005). Lopes dos Santos Santiago et al. (2011) found that there are sialidase positive and sialidase negative genotypes among G. vaginalis, It might be interesting to know whether sialidase positive G. vaginalis strains are associated more strongly with the adverse outcomes in BV. But thus far the potential link between sialidase positive G. vaginalis and biofilm formation capacity has not been elucidated. 4.4.5 Cystic Fibrosis Cystic fibrosis (CF) is an autosomal recessive genetic disorder, most common among Caucasians. It is caused by an inherited mutation in a specific chloride ion channel named the cystic fibrosis transmembrane conductance regulator (CFTR). The lack of functional CFTR molecules on the surface of mucosal tissues severely affects the production of sweat, components of the digestive juices and mucous composition. One of the most prominent features of CF is the loss of normal mucociliary clearance, resulting in extensive mucous increase in the lungs. Infecting microbes are not cleared and uncontrolled inflammation begins to cause permanent damage to the lung architecture, resulting in bronchiectasis, pulmonary hypertension and hypoxia (Peters et al. 2012). Pseudomonas aeruginosa and biofilm formation Pseudomonas aeruginosa is a key player in the pathology and morbidity of cystic fibrosis and is known to form biofilms (Singh et al. 2000). Compared with freeswimming cultures, biofilms resist clearance by the host immune system and display increased resistance to antimicrobial agents (Landry et al. 2006; Drenkard and Ausubel 2002). Furthermore, resistant strains of P. aeruginosa may be selected in the CF respiratory tract by antimicrobial therapy itself. Drenkard and Ausubel (2002) found that antibiotic-resistant phenotypic variants of P. aeruginosa with increased ability to form biofilms, arise at high frequency both in vitro and in the lungs of CF patients. Landry et al. (2006) found that P. aeruginosa biofilm development proceeds differently on surfaces coated with the glycoprotein mucin compared with biofilm 27 development on glass and surfaces coated with actin and DNA. Biofilms formed on mucin-coated surfaces developed large cellular aggregates and had increased tolerance to the antibiotic tobramycin compared with biofilms grown on glass. Furthermore, Landry et al. (2006) proposed that a specific adhesin–mucin interaction immobilizes the bacterium on the surface, resulting in a highly structured, heterogeneous biofilm that has increased tolerance to tobramycin (Laundry et al. 2006). P. aeruginosa and neuraminidase activity P. aeruginosa is an important cause of nosocomial pneumonia as well as the chief cause of lung infection in CF. Over 3 decades ago, neuraminidase production in isolates of P. aeruginosa from CF patients was described and suggested to contribute to pulmonary infection (Leprat et al. 1980). In vitro studies documented that many pulmonary pathogens including P. aeruginosa bind on asialylated glycolipids (Krivan et al. 1988), suggesting that the ability to desialylate mucosal surfaces could contribute to bacterial colonization of the airways. Analyses of P. aeruginosa gene expression in CF patients document that the PA2794 neuraminidase locus is one of the most highly expressed genes in this patient population in vivo (Lanotte et al. 2004). Unlike other respiratory pathogens, P. aeruginosa cannot use sialic acid as a carbon source, nor does it contain sialic acid as a component of its LPS (Knirel et al. 1988). Therefore it seemed likely that there was some additional function for the enzyme relevant to the pathogenesis of respiratory tract infection. Biofilm formation and neuraminidase activity Soong et al. (2006) showed that the P. aeruginosa neuraminidase is involved in biofilm formation contributing to initial colonization of the airway. Furthermore, they demonstrated that this activity can be blocked by viral neuraminidase inhibitors in clinical use indicating a novel therapeutic target for preventing bacterial pneumonia (Soong et al. 2006). 4.4.6 Propionibacterium acnes Sialidase as a possible new target in the therapy of acne vulgaris? Propionibacteria are prevalent skin colonizing bacteria with P. acnes as the most relevant one. Acne vulgaris is perhaps the most well-known skin condition caused by P. acnes, affecting up to 80% of adolescents (Percival et al. 2012). Besides 28 keratinolytic and sebosuppressive agents, antibacterial agents are an important part of the treatment of acne (Coenye et al. 2007). In cases where topical treatment is not successful or in patients at risk for scarring of the skin and pigmentary changes, isotretinoin or systemic antibiotics are indicated. Isotretinoin is widely prescribed for systemic treatment of severe acne, although the teratogenicity of isotretinoine is well documented (Coenye et al. 2007; Nakatsuji et al. 2008). In addition, several studies indicate a drastic increase in the proportion of patients carrying P. acnes strains resistant to one or more antibiotics (Coates et al. 200;, Nord & Oprica 2006; Ross et al. 2003). Although evidence for the involvement of P. acnes biofilms in the pathogenesis of acne vulgaris remains circumstantial, Coenye et al. (2007) indicate that multiple P. acnes strains can form biofilms in vitro. This could help explain the frequent failure of antimicrobial therapy in the treatment of acne. As a consequence of this, new treatments are in the running. Since the greatest concern to patients is the inflammatory stage of acne vulgaris that may lead to scarring and adverse psychological effects, Nakatsjuji et al. (2008) developed a vaccine that suppresses P. acnes induced inflammation and pathogenesis. The Nakatsjuji study (2008) revealed that sialidase-immunized mice demonstrated decreased P. acnes induced ear swelling and reduced production of the proinflammatory cytokine MIP-2. Although Nakatsjuji et al. (2008) have demonstrated that these anti-P. acnes vaccines decrease P. acnes-induced inflammation, they may not have the capability to neutralize the virulence factors secreted from P. acnes. In addition, these vaccines may lack the therapeutic effects. Another difficulty is that the anti-P. acnes vaccines have to be administrated in the early childhood. Since people cannot predict if they will suffer from acne vulgaris, many of them may be unwilling to receive these vaccins (Liu et al. 2011). As a consequence, the search for new therapeutic interventions continues. 4.5 Trypanosoma cruzi Chagas disease is an infection caused by the protozoan parasite Trypanosoma cruzi. It is an important cause of morbidity and mortality not only in endemic areas of Mexico, Central and South America but also among immigrants now residing in other areas of the world. In Chagas disease, 30% of the infected individuals ultimately develop clinically evident chronic cardiomyopathy and/or gastrointestinal 29 disease. Chronic heart disease varies widely in its manifestations, ranging from asymptomatic ECG abnormalities to congestive heart failure, arrhythmias, and/or thromboembolic events. Gastrointestinal symptoms develop in 6% of patients with Chagas disease. The most common manifestations are those related to the megasyndrome (Hemmige et al. 2012; Nagajyothi et al. 2012). Life cycle of Trypanosoma cruzi Trypanosoma cruzi has a complex life cycle involving human hosts and insect vectors as shown in Figure 6. Figure 6: Lifecycle of Trypanosoma cruzi (adopted from: http://www.dpd.cdc.gov/dpdx/HTML/TrypanosomiasisAmerican.htm) In the natural life cycle, the insect vector ingests non-dividing blood-form trypomastigotes from a mammalian host, which then transform into epimastigotes. Within 3–4 weeks, infective, non-dividing metacyclic trypomastigotes present in the hindgut of the vector are deposited with the feces of the vector during subsequent blood meals. Transmission to the new host occurs when the parasite-laden feces contaminate oral or nasal mucous membranes, the conjunctiva, and other vulnerable surfaces. The trypomastigotes enter a host cell and transform into intracellular amastigotes, which then multiply and ultimately transform into blood form trypomastigotes, which are released as the host cell ruptures. These trypomastigotes infect neighboring cells or disseminate via the lymphatics and the bloodstream and infect new cells. Although any nucleated mammalian cell can be parasitized, the cells 30 of the cardiovascular, reticuloendothelial, nervous, and muscular systems as well as adipose tissue are favored (Tanowitz et al. 1992). In addition to vector-associated transmission, the other modes of transmission include vertical transmission from mother to child, contaminated food or drink, blood transfusion and organ transplantation (Tanowitz et al. 1992). Trans-sialidase activity in T. cruzi T. cruzi expresses trans-sialidase (TS), an enzyme that transfers sialic acid from host glycoproteins to parasite glycosylphosphatidylinositol (GPI)-mucins which entirely cover the parasite surface (Buscaglia et al. 2006). Next to its location on the parasite surface, TS is also secreted into the environment (DcRubin and Schenkman 2012). The suggestion that T. cruzi has TS acitivity, arose when sialic acid was discovered in the parasite (Pereira et al. 1980). Schauer and coworkers (1983) discovered that the sialic acid composition of T. cruzi is identical to that of the host. No conventional sialic acids precursors were found in the parasite. This proposed the idea that sialic acid is transferred to the parasite from the medium (Schauer et al. 1983). In 1985, Previato and coworkers demonstrated that T. cruzi can enzymatically transfer sialic acid to itself. The presence of TS in T. cruzi was then confirmed by detecting transferase activity in trypomastigote forms (Zingales et al. 1987). Many roles of TS have been ascribed in the biology of T. cruzi and in the pathology of Chagas disease. However, the main difficulty to determine the function of TS is that knockout parasites were never obtained due to the number of copies of TS scattered through the genome and because T. cruzi does not have RNAi (DaRocha et al. 2004). The role of TS during cell invasion in Chagas disease The initial step in the establishment of Chagas disease is cell invasion. Infective T. cruzi parasites invade phagocytic and non-phagocytic cells (Burleigh and Andrews 1995). Virulent metacyclic trypomastigotes replicate inside macrophages, and although many parasites are destroyed in the phagocytic vacuole, intracellular dividing amastigotes transform into trypomastigotes that escape into the blood to infect other cells in the host. The role of TS during attachment and invasion has still not been clarified (dC Rubin and Schenkman 2012). Schenkman and coworkers (1993) found that the invasion of sialic acid deficient cells is reduced compared to wild-type cells. In addition, specific TS inhibitors were shown to reduce infection of cultured cells (Carvalho et al. 2010). 31 Treatment of cells with modified precursors of sialic acid also reduces invasion (Lieke et al. 2011). However, TS does not seem to limit cell invasion. Trypomastigotes expressing high enzymatic levels invade with similar efficiency as metacyclics, which have much less enzymatic activity (Rubin-de-Celis et al. 2006). Furthermore, overexpression of trypomastigote TS in metacyclics does not increase invasion. This indicates that sialic acid containing molecules are used for attachement and invasion without the necessity of large excess of TS activity (dCRubin and Schenkman 2012). TS and innate immunity A total of 2*10 7 sialic acid molecules are found on the surface of T. cruzi. They cause a strong negative charge. These sialic acids influence the cell invasion by obstructing both macrophage phagocytosis and complement recognition (Schenkman et al. 1994). TS secreted into the bloodstream, removes sialic acids from the platelet surface. This way, TS causes thrombocytopenia during the acute phase of Chagas disease (Tribulatti et al. 2005). An important mechanism of T. cruzi evasion is through the modulation of the immunogenic properties of dendritic cells (DCs). Infection with T. cruzi increases the number of splenic DCs. However, most splenic DCs remain immature (Chaussabel et al. 2003). The sialylated surface of T. cruzi interacts with the surface of dendritic cells. This leads to suppression in the production of the pro-inflammatory cytokine IL-12. This points out that TS has an important role in suppressing the magnitude of the innate immune responses (Erdmann et al. 2009). Intracellular roles played by TS Trypanosoma cruzi cellular invasion is characterized by the formation of a parasitophorous vacuole in the host cell. Expression of TS is highly induced when the cells become full of parasites and when amastigotes transform into trypomastigotes (Abuin et al. 1999). Therefore, it would be possible to relate the presence of large amounts of TS in the cytosol with host cell rupture. It is also possible that TS is released in the extracellular environment before cell rupture, or after host cell rupture of neighboring cells. Thus, TS may have a role in the establishment and the progression of Chagas disease. 32 TS and acquired immunity Following infection, the initial rise in parasitemia is controlled by cytokines and other mediators released by macrophages and natural killer cells. These innate immune responses are followed by a delayed polyclonal lymphocyte activation and subsequent hyper-gammaglobulinaemia triggered by TS (DosReis 1997; Rodrigues et al. 1999). During acute infection, depressed humoral and cellular immune responses coexist with a massive T and B cell polyclonal activation (Minoprio et al. 1989). In addition to the polyclonal lymphocyte activation, TS induces cell apoptosis in the thymus and peripheral ganglia (Mucci et al. 2006), exacerbates host CD4+ T lymphocyte response (Todeschini et al. 2002) and interferes with CD8+ T cell responses through changes in sialylation and interaction with host cells (Freire-deLima et al. 2010). This can contribute to immunosuppression and promotion of infection spreading. The antibodies produced by B cells, work against the catalytic site of TS. They control the levels of TS activity in serum during the acute phase of the infection (Pereira-Chioccola et al. 1994). These antibodies are mainly from IgG subclasses (Ribeirão et al. 2000). During the acute phase of the infection, T. cruzi utilizes the enormous B cell activation as a way to strengthen its resistance. Zuniga and coworkers (2002) discovered that activated B cells in Chagas disease undergo accelerated apoptosis. T. cruzi infection induces up-regulation of both Fas and Fas ligand (FasL) molecules on B cells and renders them susceptible to B cell-B cell killing. Despite inducing robust immune responses in humans, chronic infection with T. cruzi cannot be eliminated by the immune system (Martin et al. 2006). Treatment The two established medications for the treatment of Chagas disease are benznidazole and nifurtimox (Hemmige et al. 2012). Which patients benefit from pharmacological treatment remains the subject of clinical trials. Although some studies have shown the potential for benefit in treating chronic or asymptomatic Chagas disease, evidence for clear benefit is currently lacking (Hemmige et al. 2012). DNA vaccines encoding the catalytic domain of TS have been shown to induce immunity protective against systemic T. cruzi infection in mice. Giddings et al. (2010) confirmed that intranasal vaccinations with TS plus CpG induce TS-specific 33 T-cell and secretory IgA responses. The intranasal vaccination protects against conjunctival T.cruzi infection, limiting local parasite replication at the site of mucosal invasion and systemic parasite dissemination (Giddings et al. 2010). 5 Discussion Human sialidases Different studies have suggested a role of human sialidases in the development of cancer (Nishii et al. 2001; Yoshida et al. 2006). Further investigations on the exact pathological role of these sialidases could mean an alternative for the already existing techniques in the fight against cancer. Recent reports showed a higher incidence of cancer in diabetic patients than in controls. This proposes the idea that diabetes and cancer are closely related to each other in pathogenesis. Miyagi and coworkers (2008) believe that there is a great chance that NEU3 regulates common signaling pathways involved in these diseases. Extra clarification of the pathological roles of NEU3 could lead to potential applications in control of cancer and diabetes (Miyagi et al. 2008). Sialidosis en galactosialidasis, two neurodegenerative lysosomal storage diseases, are linked to dysfunctions in the NEU1 gene. Residual neuraminidase activity in patients with sialidosis and galactosialidosis is typically less than 1 percent of normal levels (d’Azzo et al. 1995). At present, there is no cure for sialidosis and galactosialidosis but more research could mean a proper solution in the future. Seyrantepe et al. (2004) showed that overexpression of Neu4 cleared storage materials from cultured fibroblasts of sialidosis and galactosialidosis patients and demonstrated that Neu4 is active against the majority of endogenous substrates of Neu1. Neu4 completely eliminated undigested substrates of Neu1 and restored the normal morphological phenotype of the lysosomal compartment, therefore offering therapeutic potential (Seyrantepe et al. 2004). However, physiological levels of Neu4 are likely not sufficient to prevent accumulation of sialylated compounds, as patients with Neu1 deficiency but with two normal Neu4 alleles still develop the disease. Therefore, it might be interesting to induce the expression of the endogenous NEU4 gene to compensate for NEU1 34 deficiency according to Seyrantepe and coworkers (2004). Maybe this could mean a breakthrough in the future. Neural cell adhesion molecules (NCAMs) are essential in neuronal network formation during development and adult synaptic plasticity (Venezo et al. 2006). Changes in the state of sialylation of NCAMs were demonstrated in certain neurological and psychiatric disorders. Wielgat et al. (2011) demonstrated that chronic stress increases PSA association with NCAM and decreases sialidase activity and causes both hippocampal atrophy and impairment of learning. It may explain the general opinion that sialidase is involved in disorders like severe depression, schizophrenia and Alzheimer disease (Kandel 2001; Gilabert-Juan et al. 2012; Mikkonen et al. 1999). Azuine et al. (2006) illustrated that sialidase dependent degradation of brain gangliosides may be responsible for the psychological and neurological impairment in the brain caused by ethanol abuse. As can be seen, sialidase activity is disrupted in these disorders. To what extent these findings will be useful for future alternative therapies is still unknown. More research and studies are needed. Changes in the sialylation state has also been discovered in epilepsy. In 2007, Isaev and coworkers demonstrated that desialylation of hippocampal slices with neuraminidase distorted the action potential threshold, delayed the onset of epileptiform activity and reduced the population spike frequency in the CA3 zone of the rat hippocampus. Because many of anti-epileptic drugs have adverse side effects and nearly one third of epilepsy patients are refractory to antiepileptic drugs, new treatment options remain a priority. Targeting negatively charged sialic acids may be an effective way to treat epilepsy (Isaeva et al. 2007). Viral, bacterial and parasitic sialidases Degradation of human sialic acids by pathogen sialidases are involved in diverse diseases. As a consequence, many efforts have been undertaken to create pharmacologically active agents. Vaccination is the most effective protection against the influenza virus. Nevertheless, because Influenza A and B viruses constantly undergo antigenic shifts, its effectiveness has been limited. As a consequence, several anti-influenza drugs have been developed. Best known are zanamivir and 35 oseltamivir which are competitive sialidase inhibitors. They hinder budding and spreading of Influenza A and B viruses, therefore reducing the duration and severity of influenza illness (Nishikawa et al. 2012). It has been known for more than a century that respiratory viruses predispose to bacterial complications. Pneumococcus is the most common agent causing community-acquired pneumonia, otitis media in children and sepsis and meningitis in adults after influenza infection. Like the influenza virus, the pneumococcus possesses sialidase activity (Simonsen 2001). Biofilm formation at the mucosal surface is important for pneumococcal colonization (Moscoso et al. 2009). NanA and NanB expression is upregulated in pneumococcal biofilms and mutants have a reduced ability to form biofilms in vitro (Trappetti et al. 2009; Parker et al. 2009). In diverse studies, early oseltamivir and zanamivir treatment in patients infected with influenza, reduced the development and severity of secondary pneumococcal infections and reduced the antibiotic use by 50 percent (Whitley et al. 2001; Treanor et al. 2000; MIST study groep 1998). Since microorganisms growing in biofilms can be up to 1000 times more resistant to antibiotics than the same free-living microorganisms, alternative therapy is urgently needed. Maybe it would be interesting to develop sialidase inhibitors specific for respiratory infections. Like the influenza virus and Streptococcus pneumoniae, Tannerella forsythia and Porphyromonas gingivalis involved in periodontitis, express sialidase activity. The sialidase of T. forsythia is involved in adhesion and invasion and also has a role in biofilm formation (Honma et al. 2011; Roy et al. 2010). Less is known about the neuraminidase in P. gingivalis, but there is increasing evidence that this neuraminidase is involved in capsule production and biofilm formation (Li et al. 2012). Roy and coworkers illustrated that sialic acid is one of the most important sources of nutrition in periodontitis and that sialic acid may serve as a receptor in the initial stage of biofilm formation. Since there is an abundance of sialic-acidcontaining glycoproteins in the oral cavity, periodontal therapy based on bacterial sialidases could serve as a possible target. BV is one of the most common vaginal infections amongst women at fertile age and can lead to spontaneous abortion, preterm delivery, low birth weight and postpartum 36 infections in pregnant women (Cauci et al. 2008). Gardnerella vaginalis, the most important pathogen in BV, expresses sialidase activity. This sialidase is involved in the downregulation of the vaginal adaptive immune system in BV. This gives an explanation for the immunosuppression which is a typical feature of BV (Cauci et al. 1998). In addition, G. vaginalis is known to produce biofilms and some genotypes produce sialidase whereas other are negative (Lopes dos Santos Santiago et al. 2011). Whether sialidase activity is involved in the process of biofilm formation, is still unknown. BV is very difficult to cure and is characterized by frequent recurrences after treatment with metronidazole or clindamycine (Bradshaw et al. 2006). It has been suggested that certain strains of lactobacilli are able to inhibit the adherence and biofilm growth of bacteria causing BV (Falagas et al. 2007). However, there still exists much controversy about their effectiveness. As a consequence, there is great interest in alternative therapies. Pseudomonas aeruginosa is a key player in the pathology and morbidity of cystic fibrosis airway infections and is known to form biofilms (Singh et al. 2000). Drenkard and Ausubel (2002) found that antibiotic-resistant phenotypic variants of P. aeruginosa with increased ability to form biofilms, arise at high frequency in the lungs of CF patients. Soong et al. (2006) showed that the P. aeruginosa neuraminidase is involved in biofilm formation contributing to initial colonization of the airway. Furthermore, they demonstrated that this activity can be blocked by viral neuraminidase inhibitors, indicating a novel therapeutic target for preventing bacterial pneumonia. As the microorganisms described above, P. acnes is also capable to produce biofilms (Coenye et al. 2007). This could help explain the frequent failure of antimicrobial therapy in the treatment of acne. Nakatsjuji et al. (2008) developed a vaccine that suppresses P. acnes induced inflammation and pathogenesis. Sialidase-immunized mice demonstrated decreased P. acnes induced ear swelling and a reduced production of the pro-inflammatory cytokine MIP-2. Unfortunately, there are some minus points. The vaccine may not have the capability to neutralize the virulence factors from P. acnes and may lack therapeutic effects. Another difficulty is that the anti-P. acnes vaccines have to be administrated in the early childhood. Since people cannot predict if they will suffer from acne vulgaris, many of them may be unwilling 37 to receive these vaccines (Liu et al. 2011). As a consequence, the search for new therapeutic interventions continues. Chagas disease, also known as American trypanosomiasis, is a potentially lifethreatening illness caused by the protozoan parasite Trypanosoma cruzi. Thirty percent of people infected, ultimately develop chronic cardiomyopathie and/or gastro-intestinal disease (Hemmige et al. 2012). T. cruzi expresses a trans-sialidase (TS), an enzyme that transfers sialic acid from the host to the surface of the parasite (Buscaglia et al. 2006). Diverse studies have demonstrated that TS plays an important role in immunosuppression, invasion and in the spread within the host (Erdmann et al. 2009; dC-Rubin and Schenkman 2012; Burleigh and Andrews 1995). The two established medications for the treatment of Chagas disease are benznidazole and nifurtimox. Although some studies have shown the potential for benefit in treating chronic or asymptomatic Chagas disease, evidence for clear benefit is currently lacking (Hemmige et al. 2012). As a result, there exists extensive research regarding prevention of Chagas Disease. DNA vaccines encoding the catalytic domain of TS have been shown to induce immunity protective against systemic T. cruzi infection in mice (Giddings et al. 2010). In this manner, TS could serve as a possible new target in vaccine development. Conclusions This paper illustrates that the role of sialidases is very diverse and ill-understood. These enzymes are not only found in humans but also in viruses, bacteria and protozoa. Within the same organism, different types of sialidases can be found. Although at the molecular level, homologies are detectable between enzymes of the mammalian families and those of bacterial, fungal and invertebrate source, a large biochemical diversity in sialidases exists (Varki and Schauer 2009). This means that species-specific sialidase medication will be needed in the treatment of different disorders. At the present, research on this subject is only carried out on laboratory animals and cell cultures with varying degrees of success. In conclusion: therapy based on sialidase-activity is still in its infancy. 38 References Abuin G, Freitas-Junior LHG, Colli W, Alves MJ et al. 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