Detection of N-glycolylneuraminic acid biomarkers in sera from patients with ovarian cancer using an engineered N-glycolylneuraminic acid-specific lectin SubB2M.

N-glycolylneuraminic acid (Neu5Gc)-containing glycans are a prominent form of aberrant glycosylation found in human tumor cells and have been proposed as cancer biomarkers. The B subunit of the subtilase cytotoxin (SubB) produced by Shiga toxigenic Escherichia coli recognises Neu5Gc containing glycans. We have previously engineered this lectin, SubB2M, for greater specificity and enhanced recognition of Neu5Gc-containing glycans. Here we further explore the utility of SubB2M to detect Neu5Gc tumor biomarkers in sera from patients with ovarian cancer. Using surface plasmon resonance (SPR) we show that SubB2M can detect the established ovarian cancer biomarker, CA125, in a highly sensitive and specific fashion in the context of human serum. These studies established conditions for screening serum samples from patients with ovarian cancer for Neu5Gc glycans. We found that serum from patients with all stages of ovarian cancer had significantly elevated mean levels of Neu5Gc glycans compared to normal controls. Serum from patients with late stage disease (stages IIIC, IV) had uniformly elevated levels of Neu5Gc glycans. Detection of Neu5Gc-glycans using SubB2M has the potential to be used as a diagnostic ovarian cancer biomarker, as well as a tool for monitoring treatment and disease progression in late stage disease.

Exploiting species specificity to understand the tropism of a human-specific toxin.

Many pathogens produce virulence factors that are specific toward their natural host. Clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) isolates are highly adapted to humans and produce an array of human-specific virulence factors. One such factor is LukAB, a recently identified pore-forming toxin that targets human phagocytes by binding to the integrin component CD11b. LukAB exhibits strong tropism toward human, but not murine, CD11b. Here, phylogenetics and biochemical studies lead to the identification of an 11-residue domain required for the specificity of LukAB toward human CD11b, which is sufficient to render murine CD11b compatible with toxin binding. CRISPR-mediated gene editing was used to replace this domain, resulting in a "humanized" mouse. In vivo studies revealed that the humanized mice exhibit enhanced susceptibility to MRSA bloodstream infection, a phenotype mediated by LukAB. Thus, these studies establish LukAB as an important toxin for MRSA bacteremia and describe a new mouse model to study MRSA pathobiology.

The pilin O-glycosylation pathway of pathogenic Neisseria is a general system that glycosylates AniA, an outer membrane nitrite reductase.

O-Glycosylation is emerging as a common posttranslational modification of surface exposed proteins in bacterial mucosal pathogens. In pathogenic Neisseria an O-glycosylation pathway modifies a single abundant protein, pilin, the subunit protein that forms pili. Here, we identify an additional outer membrane glycoprotein in pathogenic Neisseria, the nitrite reductase AniA, that is glycosylated in its C-terminal repeat region by the pilin glycosylation pathway. To our knowledge, this is the first report of a general O-glycosylation pathway in a prokaryote. We also show that AniA displays polymorphisms in residues that map to the surface of the protein. A frame-shift mutation abolishes AniA expression in 34% of Neisseria meningitidis strains surveyed, however, all Neisseria gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology.

Structure and expression in Escherichia coli K-12 of the L-asparaginase I-encoding ansA gene and its flanking regions.

Escherichia coli contains two L-asparaginase isozymes: a secreted high-affinity enzyme, L-asparaginase II (AnsII), and a low-affinity cytoplasmic enzyme, L-asparaginase I (AnsI), which is encoded by the ansA gene. The nucleotide sequence of ansA and flanking regions, comprising 2156 bp, has been determined. The ansA gene product has been identified and has a calculated Mr of 35,388; gel filtration of cell extracts indicates that the active form of the enzyme is a dimer. The deduced amino acid sequence of AnsI shows discernible similarity to AnsII in a region immediately adjacent to the proposed active-site peptide of asparaginase II as previously determined by substrate analogue binding experiments. A second open reading frame (ORF1), encoding a protein of Mr 23,336, is found 10 bp downstream from ansA; the ribosome-binding site of ORF1 overlaps the stop codon of ansA. Deletions within the 5' region of ansA abolish expression of ansA and also reduce expression of ORF1. Together, these observations suggest that ansA and ORF1 constitute an operon. A palindromic sequence exists in the 3' region of ORF1 which may function as a bidirectional transcription terminator both for the ansA-ORF1 operon and a second, convergent, ORF.

Isolation and characterization of the Neisseria meningitidis lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis.

The lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis in Neisseria meningitidis has been cloned and sequenced. By complementation of a temperature-sensitive E. coli lpxD mutant, we first cloned a meningococcal chromosomal fragment that carries the lpxD homologue. Cloning and sequence analysis of chromosomal DNA downstream of lpxD revealed the presence of the fabZ and lpxA genes. This gene cluster shows high homology to the corresponding genes from several other bacterial species. The LpxA and LpxD proteins catalyze early steps in the lipid A biosynthetic pathway, adding the O- and N-linked 3-OH fatty acyl chains, respectively. In E. coli and N. meningitidis, LpxD has the same specificity, in both cases adding 3-OH myristoyl chains; in contrast to E. coli, the meningococcal LpxA protein is presumed to add 3-OH lauroyl chains instead. The established sequence points the way to further experiments to define the basis for this difference in specificity, and should allow modification of meningococcal lipid A biosynthesis through gene exchange.

An exceptional hydroboration of substituted fluoroolefins providing tertiary alcohols.

[reaction--see text] A rare hydroboration-oxidation providing tertiary-alcohols has been achieved in the case of 1,1,2-perfluoroalkyl(aryl)ethylenes. The hydroboration of substituted perfluoroalkyl(aryl)ethylenes with dichloroborane reveals that the regioselectivity does not entirely depend on the electronics of the fluoroolefins.

The genetics of glycosylation in Gram-negative bacteria.

In recent years there has been a dramatic increase in reports of glycosylation of proteins in various Gram-negative systems including Neisseria meningitidis, Neisseria gonorrhoeae, Campylobacter jejuni, Pseudomonas aeruginosa, Escherichia coli, Caulobacter crescentus, Aeromonas caviae and Helicobacter pylori. Although this growing list contains many important pathogens (reviewed by Benz and Schmidt [Mol. Microbiol. 45 (2002) 267-276]) and the glycosylations are found on proteins important in pathogenesis such as pili, adhesins and flagella the precise role(s) of the glycosylation of these proteins remains to be determined. Furthermore, the details of the glycosylation biosynthetic process have not been determined in any of these systems. The definition of the precise role of glycosylation and the mechanism of biosynthesis will be facilitated by a detailed understanding of the genes involved.

Tetrameric repeat units associated with virulence factor phase variation in Haemophilus also occur in Neisseria spp. and Moraxella catarrhalis.

The tetrameric repeat units 5'-CAAT-3' and 5'-GCAA-3' are associated with phase variable expression of lipopolysaccharide biosynthetic genes in Haemophilus influenzae. Four other tetrameric repeat units have also been reported from H. influenzae strain Rd, 5'-CAAC-3', 5'-GACA-3', 5'-AGCT-3', and 5'-TTTA-3', which are also associated with putative virulence factors. Using oligonucleotide probes corresponding to five tandem copies of each of these tetramers, we have screened three strains of Neisseria meningitidis and one each of Neisseria gonorrhoeae, Neisseria lactamica, Haemophilus parainfluenzae, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiceptica and Moraxella catarrhalis for the presence of these motifs. We have demonstrated the presence of multiple copies of the 5'-GCAA-3' motif in all the Neisseria strains tested, and also the repeated motif 5'-CAAC-3' in M. catarrhalis. We have further demonstrated by Southern blot analysis that the 5'-CAAC-3' repeats detected in M. catarrhalis are probably associated with the same genes as in H. influenzae, but that the 5'-GCAA-3' motifs in N. meningitidis are not. The use of characterised tetrameric DNA sequences as hybridisation probes may prove useful in the identification of novel phase variable virulence determinants in organisms other than H. influenzae.

Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis.

We have identified a homologue of the adhesin AIDA-I of Escherichia coli in Neisseria meningitidis. This gene was designated nhhA (Neisseria hia homologue), as analysis of the complete coding sequence revealed that it is more closely related to the adhesins Hia and Hsf of Haemophilus influenzae. The sequence of nhhA was determined from 10 strains, and found to be highly conserved. Studies of the localisation by Western immunoblot analysis of total cell proteins and outer membrane complex preparations and by immunogold electron microscopy revealed that NhhA is located in the outer membrane. A strain survey showed that nhhA is present in 85/85 strains of N. meningitidis representative of all the major disease-associated serogroups, based on Southern blot analysis. It is expressed in the majority of strains tested by Western immunoblot.

Phase variation in meningococcal lipooligosaccharide biosynthesis genes.

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or lgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host.

Molecular analysis of the meningococcal LPS expression.

Lipopolysaccharide (LPS) is one of the major virulence factors of Neisseria meningitidis (1), with proposed roles in bacterial attachment to the host, invasion of host tissues, serum resistance, evasion of the host immune response, and the pathogenesis of sepsis syndrome. Accordingly it has become an important target for research.

The phase-variable allele of the pilus glycosylation gene pglA is not strongly associated with strains of Neisseria gonorrhoeae isolated from patients with disseminated gonococcal infection.

The Neisseria gonorrhoeae pglA gene has two alleles, one of which is phase variable. A previous study reported that all disseminated gonococcal infection (DGI) isolates contained the phase-variable allele and proposed a causal link. In the present study of 81 strains no absolute correlation between DGI and the phase-variable pglA allele was observed.

Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins.

Escherichia coli contains two L-asparaginase isozymes: L-asparaginase I, a low-affinity enzyme located in the cytoplasm, and L-asparaginase II, a high-affinity secreted enzyme. A molecular genetic analysis of the gene (ansA) encoding the former enzyme has previously been reported. We now present a molecular study of the gene, ansB, encoding L-asparaginase II. This gene was isolated by using oligonucleotide probes, whose sequences were based on the previously determined amino acid sequence. The nucleotide sequence of ansB, including 5'- and 3'-untranslated regions, was determined. The amino acid sequence of L-asparaginase II, deduced from this nucleotide sequence, contains differences at 11 positions when compared with the previously determined amino acid sequence. The deduced amino acid sequence also reveals a typical secretory signal peptide of 22 residues. A single region of sequence similarity is observed when ansA and ansB are compared. The transcriptional start site in ansB was determined, allowing the identification of the promoter region. The regulation of ansB was studied by using ansB'-'lacZ fusions, together with a deletion analysis of the 5' region upstream of the promoter. Regulation by cyclic AMP receptor protein and anaerobiosis (FNR protein) was confirmed, and the presence of nucleotide sequence motifs, with homology to cyclic AMP receptor protein and FNR protein-binding sites, investigated.

Co-dependent positive regulation of the ansB promoter of Escherichia coli by CRP and the FNR protein: a molecular analysis.

Transcription of the ansB gene, encoding L-asparaginase II, is positively regulated by cAMP receptor protein (CRP) and by the product of the fnr gene, the FNR protein. These global regulatory proteins mediate the expression of ansB in Escherichia coli in response to carbon source and to anaerobiosis, respectively, and are required concurrently for optimal ansB expression. The mechanism whereby CRP and FNR interact co-operatively with the ansB promoter to achieve transcription has not previously been established. We have utilized an ansB'-'lacZ fusion, in conjunction with deletion analysis and site-directed mutagenesis, to identify two sites which interact with these regulatory proteins in the ansB promoter. The first is an FNR site, centred 41.5 bp upstream of the major transcriptional start site. The second site, located 28 bp upstream of the FNR site, is the site of CRP regulation. This site is homologous to both the CRP and FNR binding-site consensus sequences and may respond to both CRP and FNR. The concurrent requirement for CRP and FNR for optimal expression of ansB may be explained if, first, essentially no transcription occurs unless the FNR is bound at the downstream site, and, second, the level of transcription when FNR alone is present is enhanced when CRP binds at the upstream site.

Tandem repeats of the tetramer 5'-CAAT-3' present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae.

A novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase-variable LPS structure expressed by Haemophilus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neisseria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)(n) encoded by multiple tandem repeats of 5'-CAAT-3'. This observation suggested that (SINQ)(n) might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5'-CAAT-3' repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine-SDS-PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)(n) is not a prerequisite for the enzymatic function of Lic2A and that the 5'-CAAT-3' repeats in lic2A function solely as a mechanism for generating phase variation. This observation suggested that wide variation in the number of 5'-CAAT-3' repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5'-CAAT-3' repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)(n) indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.

Cloning and molecular analysis of the Salmonella enterica ansP gene, encoding an L-asparagine permease.

A gene (ansP), which encodes an L-asparagine permease, has been isolated from a cosmid library of Salmonella enterica during screening for recombinant clones which encode L-asparaginase. Nucleotide sequence analysis reveals that the gene product is a polypeptide of 497 amino acid residues, containing 12 putative transmembrane segments. The calculated molecular mass is 54 kDa, although maxicell analysis by SDS-PAGE gave an apparent molecular mass of 37 kDa. Comparison of the deduced amino acid sequence with sequence databases showed significant homology with a family of basic and aromatic amino acid permeases. Strains containing the cloned ansP gene demonstrated a many-fold increase in L-asparagine uptake in comparison with control strains.

A facile synthesis of perfluoroalkyl vinyl iodides and their palladium-mediated cross-coupling reactions.

Catalytic amounts of zinc, as low as 10 mol %, in the presence of trifluoroacetic acid (TFA) initiate the radical addition of perfluoroalkyl iodides to terminal alkynes with high regio- and stereoselectivities. Palladium-mediated cross-coupling of these (E)-perfluoroalkyl vinyl iodides allows for a facile synthesis of potentially useful fluoroorganic intermediates.

Regulation of the ansB gene of Salmonella enterica.

The expression of L-asparaginase II (encoded by ansB) in Salmonella enterica was found to be positively regulated by the cAMP receptor protein (CRP) and anaerobiosis. The anaerobic regulation of the S. enterica ansB gene is not mediated by the anaerobic transcriptional activator FNR. This is unlike the situation of the ansB gene of Escherichia coli, which is dependent on both CRP and FNR. To investigate this fundamental difference in the regulation of L-asparaginase II expression in S. enterica, the ansB gene was cloned and the nucleotide sequence of the promoter region determined. Sequence analysis and transcript mapping of the 5' promoter region revealed a single transcriptional start point (tsp) and two regulatory sites with substantial homology with those found in E. coli. One site, centred -90.5 bp from the tsp, is homologous to a hybrid CRP/FNR ('CF') site which is the site of CRP regulation in the E. coli promoter. The other site, centred 40.5 bp upstream of the tsp, is homologous to the FNR binding site of the E. coli promoter. Significantly, however, a single base-pair difference exists in this site, at a position of the related CRP and FNR DNA-binding site consensus sequences known to be involved in CRP versus FNR specificity. Site-directed mutagenesis indicates that this single difference, relative to the homologous E. coli site, results in a CRP binding site and the observed FNR-independent ansB expression in S. enterica.(ABSTRACT TRUNCATED AT 250 WORDS)