Andrea Maggioni

Molecular and Genomic Analysis of Genes Encoding Surface-Anchored Proteins from Clostridium difficile

ABSTRACT The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79–685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79–685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins.

Revisiting the role of histo-blood group antigens in rotavirus host-cell invasion

Histo-blood group antigens (HBGAs) have been proposed as rotavirus receptors. H type-1 and Lewis(b) antigens have been reported to bind VP8* from major human rotavirus genotypes P[4], P[6] and P[8], while VP8* from a rarer P[14] rotavirus recognizes A-type HBGAs. However, the role and significance of HBGA receptors in rotavirus pathogenesis remains uncertain. Here we report that P[14] rotavirus HAL1166 and the related P[9] human rotavirus K8 bind to A-type HBGAs, although neither virus engages the HBGA-specific α1,2-linked fucose moiety. Notably, human rotaviruses DS-1 (P[4]) and RV-3 (P[6]) also use A-type HBGAs for infection, with fucose involvement. However, human P[8] rotavirus Wa does not recognize A-type HBGAs. Furthermore, the common human rotaviruses that we have investigated do not use Lewis(b) and H type-1 antigens. Our results indicate that A-type HBGAs are receptors for human rotaviruses, although rotavirus strains vary in their ability to recognize these antigens.

Structure and function of nucleotide sugar transporters: Current progress

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure–function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

Binding of a natural anthocyanin inhibitor to influenza neuraminidase by mass spectrometry

The binding of a natural anthocyanin to influenza neuraminidase has been studied employing mass spectrometry and molecular docking. Derived from a black elderberry extract, cyanidin-3-sambubiocide has been found to be a potent inhibitor of sialidase activity. This study reveals the molecular basis for its activity for the first time. The anthocyanin is shown by parallel experimental and computational approaches to bind in the so-called 430-cavity in the vicinity of neuraminidase residues 356–364 and 395–432. Since this antiviral compound binds remote from Asp 151 and Glu 119, two residues known to regulate neuraminidase resistance, it provides the potential for the development of a new class of antivirals against the influenza virus without this susceptibility.

Characterization of surface layer proteins from Clostridium difficile by liquid chromatography/electrospray ionization mass spectrometry

Surface layers (S-layers) are regularly ordered protein subunits found as the outermost cell envelope component of many bacteria. Most S-layers are composed of a single protein or glycoprotein species with a molecular weight varying between 40 and 200 kDa. Clostridium difficile is the most common cause of antibiotic associated diarrhea (AAD) and pseudomembranous colitis (PMC) in humans. Detection of the S-layer in some C. difficile strains, and preliminary characterization of two glycoproteins, P36 and P47, involved in the composition of the S-layer of one of these strains (C. difficile C253), led us to investigate the most appropriate conditions for purification and chemical characterization of these proteins. This work describes the results obtained when liquid chromatography (LC) coupled to mass spectrometry (MS) using electrospray ionization was applied to the analysis of C. difficile S-layer proteins (SLPs). In this way the molecular weights of the two SLP components, P36 and P47, were detected to be 34,258 +/- 2 and 39,545 +/- 3 Da, respectively. These data deviate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results by 1.85 and 7.5 kDa. To confirm the LC-MS results, an alternative molecular weight analysis was performed: the two S-layer proteins were isolated by semipreparative high performance liquid chromatography (HPLC), concentrated, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). The two SLP subunits were digested with protease V8, and the peptide maps were determined by LC-MS using a C18 column. Finally, preliminary results about peptide glycosylation were obtained.

Detection of Ligand Binding to Nucleotide Sugar Transporters by STD NMR Spectroscopy

Fifty percent of all drugs on the market target integral membrane proteins, however, crystallization and structure determination of these proteins for use in the drug discovery process, particularly for highly hydrophobic multiple-membranespanning proteins, has proven problematic. In fact, integral membrane proteins represent less than 1% of all available crystal structures. The hydrophobic nature and poor solubility of multiple-membrane-spanning proteins also complicates protein–ligand interaction studies. A much-used approach involves generating recombinant soluble forms of the catalytic/ receptor domain. However, this approach is only effective when the catalytic/receptor domain functions independently of the membrane. In these instances, the contribution of the membrane environment in protein function is ignored. Ligand interactions with membrane-anchored proteins, such as the interaction of binding ligands to the human sweet receptor, aIIbb3 integrin, which was reconstituted in proteoliposomes [3]

Characterisation of CMP-Sialic Acid Transporter Substrate Recognition

CMP-sialic acid transporter: We report an in-depth, multidisciplinary, structural study that has identified the amino acid residues intimately involved in CMP-sialic acid transporter (CST) substrate specificity. Our data provide a significant contribution towards a better understanding the structure-function relationship of this important family of transporters and the rational design of CST inhibitors.

Novel 3,4-disubstituted-Neu5Ac2en derivatives as probes to investigate flexibility of the influenza virus sialidase 150-loop

Novel 3,4-disubstituted-Neu5Ac2en derivatives have been synthesised to probe the open 150-loop conformation of influenza virus sialidases. Both equatorially and axially (epi) substituted C4 amino and guanidino 3-(p-tolyl)allyl-Neu5Ac2en derivatives were prepared, via the 4-epi-hydroxy derivative. The equatorially-substituted 4-amino derivative showed low micromolar inhibition of both group-1 (pdm09 H1N1) and group-2 (pdm57 H2N2) sialidases, and provides the first in vitro evidence that a group-2 sialidase may exhibit 150-loop flexibility.

Direct investigation of the Aspergillus GDP-mannose transporter by STD NMR spectroscopy.

Aspergillus species, predominantly A. fumigatus and A. flavus, represent some of the most important air-borne fungal pathogens world-wide. Found ubiquitously in nature, Aspergillus spp. cause a variety of diseases depending on the immune status of the host. Most cases of invasive aspergillosis (IA) are associated with HIV infections, haematological malignancies, leukaemia or lymphoma. The harmless status of Aspergillus has changed over the past two decades, with the frequency of IA increasing 14-fold in the past ten years. IA patients have a mortality of over 95%, and the fungus is able to cause several forms of disease in humans of which IA is the most severe. The high mortality rate of this disease warrants increased efforts towards understanding the basic principles of Aspergillus pathogenicity. New antifungal agents have aided in both the treatment of fungal infections and in the prevention of disease in susceptible individuals, but resistance is an increasing concern. Only a few potential antifungal targets have been exploited to date and there is a critical need for the discovery and development of novel antifungal agents that will result in improved therapy in this ever-expanding patient population. A novel class of antifungal agents called the echinocandins target the synthesis of b-(1,3)-glucan, the a major cell wall component (galactomannan, Figure 1A). Galactomannan is composed of a linear mannan core branched with short 1,5linked galactofuranose chains, bound covalently to the cell wall b-(1,3)-glucan, anchored to the lipid membrane by a glycosylphosphatidylinositol (GPI) anchor, or released in the environment during tissue invasion or growth in culture. 8] Besides being an abundant component of the extracellular matrix, secreted galactomannans are a serological diagnostic of IA. The efficient biosynthesis of the fungus galactomannan cell wall requires the transport of nucleotide sugars, the donor substrates for glycosyltransferases, from the cytosol into the Golgi lumen. This critical task is carried out by a group of proteins termed nucleotide-sugar transporters (NSTs). Some NST activities are found in all organisms, however some are restricted to certain lineages. For example, a Golgi resident GDP-mannose (GDP-Man) transporter (Figure 1B) is not found in mammalian cells but in yeast, Leishmania, and Aspergillus. Since NST activity is required prior to glycosylation, there has been great interest in better understanding NST specificity, regulation, and cellular targeting. At present, there is relatively little information on the structure of the NST protein complex, the basis for NST specificity, and the targeting and organization of NSTs within the cell. Loss of expression of all Man-containing molecules in A. fumigatus by a targeted deletion of the genes involved in GDP-Man biosynthesis resulted in complete loss of virulence in macrophages and susceptible mice. In addition, a more recent study has shown that the GDP-Man transporter (encoded by the gmtA gene) is essential in Aspergillus niger. Therefore, the Aspergillus GDP-Man transporter (AspGMT), which is not found in mammalian cells, represents a promising drug target for the development of novel anti-fungal drugs that inhibit GDP-Man translocation into the Golgi lumen required for the biosynthesis of the fungal virulence factor galactomannan. NSTs are Golgior endoplasmic reticulum (ER)-resident type III transmembrane proteins with 8–10 predicted membranespanning domains. There is currently no 3Dor 2D-crystal Figure 1. A) The cell wall of Aspergillus sp. is composed of b-(1,3)-glucan, chitin and galactomannan. Galactomannan is covalently attached to b-(1,3)glucan or anchored to the lipid membrane through a GPI anchor. B) The biosynthesis of the fungus galactomannan cell wall requires the transport of GDP-Man, the donor substrates for mannosyltransferases, from the cytosol into the Golgi lumen. GDP-Man transported into the Golgi is coupled with the export of GMP in an antiporter mechanism.

The Targeted Expression of Nucleotide Sugar Transporters to the E. coli Inner Membrane

The heterologous expression of functional mammalian integral membrane proteins still represents a significant hurdle towards evaluating the relationship between their structure and function. We have therefore utilised the OmpA signal sequence to deliberately target the expression of a mammalian nucleotide sugar transporter, the murine CMP-sialic acid transporter, to the E. coli inner membrane. The functionality of the recombinant CMP-sialic acid transporter could then be evaluated either following the spheroplasting of E. coli cells or through the isolation of the E. coli inner membrane and the formation of mixed phosphatidylcholine-inner membrane proteoliposomes.