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1.  Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage, Sf101, from a serotype 7a strain 
BMC Genomics  2014;15(1):742.
Shigella flexneri is the major cause of bacillary dysentery in the developing countries. The lipopolysaccharide (LPS) O-antigen of S. flexneri plays an important role in its pathogenesis and also divides S. flexneri into 19 serotypes. All the serotypes with an exception for serotype 6 share a common O-antigen backbone comprising of N-acetylglucosamine and three rhamnose residues. Different serotypes result from modification of the basic backbone conferred by phage-encoded glucosyltransferase and/or acetyltransferase genes, or plasmid-encoded phosphoethanolamine transferase. Recently, a new site for O-acetylation at positions 3 and 4 of RhaIII, in serotypes 1a, 1b, 2a, 5a and Y was shown to be mediated by the oacB gene. Additionally, this gene was shown to be carried by a transposon-like structure inserted upstream of the adrA region on the chromosome.
In this study, a novel bacteriophage Sf101, encoding the oacB gene was isolated and characterised from a serotype 7a strain. The complete sequence of its 38,742 bp genome encoding 66 open reading frames (orfs) was determined. Comparative analysis revealed that phage Sf101 has a mosaic genome, and most of its proteins were >90% identical to the proteins from 12 previously characterised lambdoid phages. In addition, the organisation of Sf101 genes was found to be highly similar to bacteriophage Sf6. Analysis of the Sf101 OacB identified two amino acid substitutions in the protein; however, results obtained by NMR spectroscopy confirmed that Sf101-OacB was functional. Inspection of the chromosomal integration site of Sf101 phage revealed that this phage integrates in the sbcB locus, thus unveiling a new site for integration of serotype-converting phages of S. flexneri, and determining an alternative location of oacB gene in the chromosome. Furthermore, this study identified oacB gene in several serotype 7a isolates from various regions providing evidence of O-acetyl modification in serotype 7a.
This is the first report on the isolation of bacteriophage Sf101 which contains the S. flexneri O-antigen modification gene oacB. Sf101 has a highly mosaic genome and was found to integrate in the sbcB locus. These findings contribute an advance in our current knowledge of serotype converting phages of S. flexneri.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-742) contains supplementary material, which is available to authorized users.
PMCID: PMC4159516  PMID: 25174528
Shigella flexneri; Bacteriophage; O-antigen modification; Serotype-conversion
2.  Genetic and structural identification of an O-acyltransferase gene (oacC) responsible for the 3/4-O-acetylation on rhamnose III in Shigella flexneri serotype 6 
BMC Microbiology  2014;14:266.
O-antigen (O-polysaccharide) of the lipopolysaccharide is a highly variable cell component of the outer membrane in Shigella flexneri. It defines the serospecificity and plays an important role in the pathogenesis of shigellosis. There are two distinct O-antigen forms for the 19 serotypes of S. flexneri: one for serotypes 1–5, X, Y, 7 (and their subtypes), and the other for serotype 6. Although having different basal O-polysaccharide structures, the two forms share a common disaccharide fragment [→2)-α-l-RhapIII-(1 → 2)-α-l-RhapII]. In serotype 6 and some non-6 serotypes, RhaIII is O-acetylated at position either 3 or 4 (3/4-O-acetylation), conferring to the hosts a novel antigenic determinant named O-factor 9. An acyltransferase gene (oacB) responsible for this modification has been identified in serotypes 1a, 1b, 2a, 5a, and Y, but not in serotype 6.
Using genetic, serological, and chemical approaches, another acyltransferase gene named oacC was demonstrated to be responsible for the 3/4-O-acetylation on RhaIII in the O-antigen of S. flexneri serotype 6. Inactivation of the oacC gene resulted in the loss of the 3/4-O-acetyltion, and the cloned oacC gene restored this modification upon transformation. In accordance with the similarity in the acceptor substrate structure and high sequence homology (72% identity) between oacC and oacB, oacC has the interchangeable function with the oacB gene in mediation of the 3/4-O-acetylation. The oacC gene is located in a prophage on the chromosome and presented in all 77 serotype 6 strains tested.
Identification and functional characterization of the O-acetyltransferase encoding gene, oacC, shows that it is involved in O-antigen modification by 3/4-O-acetylation on RhaIII specific to serotype 6.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-014-0266-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4206707  PMID: 25327486
Shigella flexneri; 3/4-O-acetylation; Acyltransferase; oacC; O-antigen; Anti-O-factor 9 serum
3.  Identification of a divergent O-acetyltransferase gene oac1b from Shigella flexneri serotype 1b strains 
Shigella flexneri is a leading cause of bacterial dysentery in developing countries. Among the 15 known serotypes, four (1b, 3a, 3b and 4b) contain a group 6 epitope due to an acetyl group connected to the O-2 position of rhamnose III on the tetrasaccharide structure of the lipopolysaccharide. O-acetyltransferase encoded by a bacteriophage, Sf6, mediates the acetylation reaction. We found that the oac gene in serotype 1b strains was very different from that in serotypes 3a, 3b and 4b strains and is herein after referred to as oac1b which shares with oac 88%–89% identity at the DNA level and 85% identity at the protein level. Considering that S. flexneri strains of serotypes 1–5 share a recent common ancestry, the divergent oac1b is more likely to have been obtained from outside S. flexneri than to have undergone rapid divergence from the oac gene in the other serotypes (3a, 3b and 4b) within S. flexneri. The cloned oac1b gene was found to perform the same acetylation function as oac. Analysis of the genomic regions flanking oac1b showed that it was present in a prophage on the chromosome and the organizational structure is different from that of phage Sf6. Additionally, phage conversion assay showed that serotype 1b cannot be generated by infecting serotype 1a strains with Sf6. We conclude that oac1b was carried by a non-Sf6 phage and is uniquely present in serotype 1b.
PMCID: PMC3630932  PMID: 26038428
O-acetyltransferase; serotype; serotype 1b; Shigella flexneri
4.  Serotype-Converting Bacteriophage SfII Encodes an Acyltransferase Protein That Mediates 6-O-Acetylation of GlcNAc in Shigella flexneri O-Antigens, Conferring on the Host a Novel O-Antigen Epitope 
Journal of Bacteriology  2014;196(20):3656-3666.
Shigella flexneri O-antigen is an important and highly variable cell component presented on the outer leaflet of the outer membrane. Most Shigella flexneri bacteria share an O-antigen backbone composed of →2)-α-l-RhapIII-(1→2)-α-l-RhapII-(1→3)-α-l-RhapI-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various chemical groups to different sugars, giving rise to diverse O-antigen structures and, correspondingly, to various serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on RhaI or/and RhaIII, and phosphorylation with phosphoethanolamine on RhaII or/and RhaIII. Recently, a new O-antigen modification, namely, O-acetylation at position 6 of N-acetylglucosamine (GlcNAc), has been identified in S. flexneri serotypes 2a, 3a, Y, and Yv. In this study, the genetic basis of the 6-O-acetylation of GlcNAc in S. flexneri was elucidated. An O-acyltransferase gene designated oacD was found to be responsible for this modification. The oacD gene is carried on serotype-converting bacteriophage SfII, which is integrated into the host chromosome by lysogeny to form a prophage responsible for the evolvement of serotype 2 of S. flexneri. The OacD-mediated 6-O-acetylation also occurs in some other S. flexneri serotypes that carry a cryptic SfII prophage with a dysfunctional gtr locus for type II glucosylation. The 6-O-acetylation on GlcNAc confers to the host a novel O-antigen epitope, provisionally named O-factor 10. These findings enhance our understanding of the mechanisms of the O-antigen variation and enable further studies to understand the contribution of the O-acetylation to the antigenicity and pathogenicity of S. flexneri.
PMCID: PMC4187690  PMID: 25112477
5.  Serological Identification and Prevalence of a Novel O-Antigen Epitope Linked to 3- and 4-O-Acetylated Rhamnose III of Lipopolysaccharide in Shigella flexneri 
Journal of Clinical Microbiology  2014;52(6):2033-2038.
Shigella flexneri is the major cause of shigellosis in developing countries. All serotypes except for serotype 6 share an O-antigen backbone composed of a →2)-α-l-RhapIII-(1→2)-α-l-RhapII-(1→3)-α-l-RhapI-(1→3)-β-d-GlcpNAc-(1→ tetrasaccharide repeat. It can be modified by the addition of a glucosyl group to one or more sugar residues and/or an O-acetyl group to RhaI and/or a phosphoethanolamine to RhaII and/or RhaIII. These modifications give rise to type I-, IC-, II-, IV-, and V- and to group 6-, 7,8-, and MASF IV-1-specific antigenic determinants, which comprise the current serotyping scheme of S. flexneri. Recently, another O-antigen modification created by adding an O-acetyl group to RhaIII at position 3 or 4 (3/4-O-acetylation) has been found in S. flexneri serotypes 1a, 1b, 2a, 5a, Y, and 6. A new O-acyltransferase gene named oacB has been shown to mediate the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not in 6. In this work, we studied the distribution of the 3/4-O-acetylation in S. flexneri and the antigenicity that resulted from this modification. PCR screening of the oacB gene in clinical isolates of S. flexneri demonstrated that the oacB-mediated 3/4-O-acetylation is widespread in serotypes 1a, 1b, 2a, 5a, and Y. Serological analysis indicated that this modification confers the host with a novel antigenic determinant that is provisionally named group O factor 9. These findings enhance our understanding of the varieties of O-antigenic determinants related to O-antigen modification in S. flexneri and will assist epidemiological studies and vaccine development.
PMCID: PMC4042796  PMID: 24671799
6.  Identification of an O-Acyltransferase Gene (oacB) That Mediates 3- and 4-O-Acetylation of Rhamnose III in Shigella flexneri O Antigens 
Journal of Bacteriology  2014;196(8):1525-1531.
O antigen (O polysaccharide) is an important and highly variable cell component present on the surface of cells which defines the serospecificity of Gram-negative bacteria. Most O antigens of Shigella flexneri, a cause of shigellosis, share a backbone composed of →2)-α-l-RhapIII-(1→2)-α-l-RhapII-(1→3)-α-l-RhapI-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various substituents, giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on RhaI, and phosphorylation with phosphoethanolamine on RhaII or/and RhaIII. Recently, two new O-antigen modifications, namely, O-acetylation at position 3 or 4 of RhaIII and position 6 of GlcNAc, have been identified in several S. flexneri serotypes. In this work, the genetic basis for the 3/4-O-acetylation on RhaIII was elucidated. Bioinformatic analysis of the genome of S. flexneri serotype 2a strain Sf301, which carries 3/4-O-acetylation on RhaIII, revealed an O-acyltransferase gene designated oacB. Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not serotype 6, which has a different O-antigen backbone structure. The oacB gene is carried by a transposon-like structure located in the proA-adrA region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in S. flexneri. These findings enhance our knowledge of S. flexneri O-antigen modifications and shed light on the origin of new O-antigen variants.
PMCID: PMC3993362  PMID: 24509314
7.  Evaluation of De-O-Acetylated Meningococcal C Polysaccharide-Tetanus Toxoid Conjugate Vaccine in Infancy: Reactogenicity, Immunogenicity, Immunologic Priming, and Bactericidal Activity against O-Acetylated and De-O-Acetylated Serogroup C Strains 
Infection and Immunity  2001;69(4):2378-2382.
The polysaccharide capsule of serogroup C Neisseria meningitidis (MenC) has been integral to vaccine development. Licensed MenC vaccines contain the O-acetylated (OAc+) form of polysaccharide. Some MenC strains have de-O-acetylated (OAc−) polysaccharide, which may affect antibody specificity and functional activity when used in a vaccine. We evaluated an OAc-MenC conjugate-tetanus toxoid conjugate (MCC-TT) vaccine given concomitantly with whole-cell diphtheria-tetanus-pertussis, Haemophilus influenzae type b, and oral polio immunization in 83 infants at 2, 3, and 4 months of age. Serum bactericidal activities (SBA) against OAc+ and OAc− MenC strains and OAc+ and OAc− polysaccharide-specific immunoglobulin G (IgG) levels were evaluated. MCC-TT vaccine was well tolerated. All infants produced SBA titers of ≥8 after a single dose at 2 months of age. The SBA geometric mean titer for OAc+ strain C11 increased from 2.7 (95% confidence interval [CI] 2.2 to 3.2) to 320 (95% CI, 237 to 432), 773 (95% CI, 609 to 982), and 1,063 (95% CI, 856 to 1319) after one, two, and three doses of MCC-TT, respectively. OAc− IgG levels were twice as high as OAc+ IgG levels after the primary series of MCC-TT vaccine, and the SBA was significantly higher against the OAc− MenC strain. Antibody responses to booster vaccination with either OAc+ MenC polysaccharide vaccine (MACP) or a fourth dose of MCC-TT at 14 months of age provided evidence of immunologic memory. The acetylation status of the booster vaccine influenced the specificity of the response, with significantly higher OAc− IgG levels and SBA after MCC-TT vaccine compared to MACP vaccine but similar OAc+ antibody levels. MCC-TT vaccine is highly immunogenic and primes for immunologic memory against OAc+ and OAc− MenC strains in infancy.
PMCID: PMC98168  PMID: 11254596
8.  Spectrum of Pneumococcal Serotype 11A Variants Results from Incomplete Loss of Capsule O-Acetylation 
Journal of Clinical Microbiology  2014;52(3):758-765.
Streptococcus pneumoniae is a significant bacterial pathogen that expresses >90 capsule serotypes. Conventional serotyping methods assume that each serotype is a genetically and antigenically distinct entity; however, recent investigations have revealed pneumococcal isolates that cannot be unambiguously serotyped because they share the properties of more than one serotype. Here, we employed a novel serotyping method and NMR spectroscopy to examine clinical isolates sharing properties of serotypes 11A and 11E. These ambiguous clinical isolates were provisionally named 11A variant (11Av) isolates. Serotype 11A pneumococci characteristically express capsule β-galactose-6-O-acetylation (βGal6OAc) mediated by the capsule synthesis gene wcjE, while 11E strains contain loss-of-function mutations in wcjE and completely lack the expression of βGal6OAc. Although 11Av isolates also contained mutated wcjE alleles, 11Av clinical isolates were composed of antigenically homogeneous bacteria expressing reduced amounts of 11A-specific capsule antigen. NMR data confirmed reduced but detectable amounts of βGal6OAc on 11Av capsule polysaccharide. Furthermore, the transformation of strains with wcjE alleles from 11Av strains was sufficient to restore partial βGal6OAc in an 11E background. We conclude that, instead of being distinct entities, serotypes 11A and 11E represent two extremes of an antigenic spectrum resulting from variable capsule O-acetylation secondary to heterologous wcjE mutations. These findings challenge whether all clinically relevant pneumococci can be definitively categorized into distinct serotypes.
PMCID: PMC3957789  PMID: 24352997
9.  Burkholderia thailandensis oacA Mutants Facilitate the Expression of Burkholderia mallei-Like O Polysaccharides▿  
Infection and Immunity  2010;79(2):961-969.
Previous studies have shown that the O polysaccharides (OPS) expressed by Burkholderia mallei are similar to those produced by Burkholderia thailandensis except that they lack the 4-O-acetyl modifications on their 6-deoxy-α-l-talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designated oacA, expressed by B. thailandensis that accounts for this phenomenon. Utilizing the B. thailandensis and B. mallei lipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by the oacA mutant, B. thailandensis ZT0715, were antigenically similar to those produced by B. mallei ATCC 23344. In addition, immunoblot analyses demonstrated that when B. mallei ATCC 23344 was complemented in trans with oacA, it synthesized B. thailandensis-like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by the oacA mutant reacted strongly with the B. mallei LPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4-O acetylation and 2-O methylation of B. thailandensis OPS antigens and that ZT0715 may provide a safe and cost-effective source of B. mallei-like OPS to facilitate the synthesis of glanders subunit vaccine candidates.
PMCID: PMC3028842  PMID: 21115721
10.  Development of a Multiplex PCR Assay Targeting O-Antigen Modification Genes for Molecular Serotyping of Shigella flexneri ▿  
Journal of Clinical Microbiology  2011;49(11):3766-3770.
Shigella flexneri is the major Shigella species that causes diarrheal disease in developing countries. It is further subdivided into 15 serotypes based on O-antigen structure. Serotyping of S. flexneri is important for epidemiological purposes. In this study, we developed a multiplex PCR assay targeting the O-antigen synthesis gene wzx and the O-antigen modification genes gtrI, gtrIC, gtrII, oac, gtrIV, gtrV, and gtrX for molecular serotyping of S. flexneri. The multiplex PCR assay contained eight sets of specific PCRs in a single tube and can identify 14 of the 15 serotypes (the exception being serotype Xv) of S. flexneri recognized thus far. A nearly perfect concordance (97.8%) between multiplex PCR assay and slide agglutination was observed when 358 S. flexneri strains of various serotypes were analyzed, except that 8 strains were carrying additional cryptic and/or defective serotype-specific genes. The multiplex PCR assay provides a rapid and specific method for the serotype identification of S. flexneri.
PMCID: PMC3209073  PMID: 21880974
11.  Draft genome sequences of the type strains of Shigella flexneri held at Public Health England: comparison of classical phenotypic and novel molecular assays with whole genome sequence 
Gut Pathogens  2014;6:7.
Public Health England (PHE) holds a collection of Shigella flexneri Type strains isolated between 1949 and 1972 representing 15 established serotypes and one provisional type, E1037. In this study, the genomes of all 16 PHE Type strains were sequenced using the Illumina HiSeq platform. The relationship between core genome phylogeny and serotype was examined.
The most common target gene for the detection of Shigella species in clinical PCR assays, ipaH, was detected in all genomes. The type-specific target genes were correctly identified in each genome sequence. In contrast to the S. flexneri in serotype 5 strain described by Sun et al. (2012), the two PHE serotype 5 Type strains possessed an additional oac gene and were differentiated by the presence (serotype 5b) or absence (serotype 5a) of gtrX. The somatic antigen structure and phylogenetic relationship were broadly congruent for strains expressing serotype specific antigens III, IV and V, but not for those expressing I and II. The whole genome phylogenies of the 15 isolates sequenced showed that the serotype 6 Type Strain was phylogenetically distinct from the other S. flexneri serotypes sequenced. The provisional serotype E1037 fell within the serotype 4 clade, being most closely related to the Serotype 4a Type Strain.
The S. flexneri genome sequences were used to evaluate phylogenetic relationships between Type strains and validate genotypic and phenotypic assays. The analysis confirmed that the PHE S. flexneri Type strains are phenotypically and genotypically distinct. Novel variants will continue to be added to this archive.
PMCID: PMC3972513  PMID: 24684748
Shigella flexneri type strains; Next generation sequencing technology; Molecular serotyping
12.  Flow-cytometric monitoring of disease-associated expression of 9-O-acetylated sialoglycoproteins in combination with known CD antigens, as an index for MRD in children with acute lymphoblastic leukaemia: a two-year longitudinal follow-up study 
BMC Cancer  2008;8:40.
Over expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs, abbreviated as OAcSGP) has been demonstrated as a disease-associated antigen on the lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Achatinin-H, a lectin, has selective affinity towards terminal 9-O-acetylated sialic acids-α2-6-Nacetylated galactosamine. Exploring this affinity, enhanced expression of OAcSGP was observed, at the onset of disease, followed by its decrease with chemotherapy and reappearance with relapse. In spite of treatment, patients retain the diseased cells referred to as minimal residual disease (MRD) responsible for relapse. Our aim was to select a suitable template by using the differential expression of OAcSGP along with other known CD antigens to monitor MRD in peripheral blood (PB) and bone marrow (BM) of Indian patients with B- or T-ALL during treatment and correlate it with the disease status.
A two-year longitudinal follow-up study was done with 109 patients from the onset of the disease till the end of chemotherapy, treated under MCP841protocol. Paired samples of PB (n = 1667) and BM (n = 999) were monitored by flow cytometry. Three templates selected for this investigation were OAcSGP+CD10+CD19+ or OAcSGP+CD34+CD19+ for B-ALL and OAcSGP+CD7+CD3+ for T-ALL.
Using each template the level of MRD detection reached 0.01% for a patient in clinical remission (CR). 81.65% of the patients were in CR during these two years while the remaining relapsed. Failure in early clearance of lymphoblasts, as indicated by higher MRD, implied an elevated risk of relapse. Soaring MRD during the chemotherapeutic regimen predicted clinical relapse, at least a month before medical manifestation. Irrespective of B- or T-lineage ALL, the MRD in PB and BM correlated well.
A range of MRD values can be predicted for the patients in CR, irrespective of their lineage, being 0.03 ± 0.01% (PB) and 0.05 ± 0.015% (BM). These patients may not be stated as normal with respect to the presence of MRD. Hence, MRD study beyond two-years follow-up is necessary to investigate further reduction in MRD, thereby ensuring their disease-free survival. Therefore, we suggest use of these templates for MRD detection, during and post-chemotherapy for proper patient management strategies, thereby helping in personalizing the treatment.
PMCID: PMC2268943  PMID: 18241334
13.  Expression of neuroepithelial transforming gene 1 is enhanced in oesophageal cancer and mediates an invasive tumour cell phenotype 
Neuroepithelial Transforming Gene 1 (NET1) is a well characterised oncoprotein and a proven marker of an aggressive phenotype in a number of cancers, including gastric adenocarcinoma. We aimed to investigate whether NET1 plays a functional role in oesophageal cancer (OAC) and its pre-malignant phenotype Barrett’s oesophagus.
Baseline NET1 mRNA levels were determined by qPCR across a panel of six cell lines, including normal oesophageal, Barrett’s and OAC derived cells. Quantification of NET1 protein in OAC cells was performed using Western blot and immunofluorescence. NET1 expression was modulated by treating with lysophosphatidic acid (LPA) and NET1-specific siRNA. The functional effects of NET1 knockdown were assessed in vitro using proliferation, migration and invasion assays.
NET1 expression was increased in Barrett’s and in OAC-derived cells in comparison to normal oesophageal cells. The highest expression was observed in OE33 a Barrett’s-related OAC cell line. NET1 protein and mRNA expression was enhanced by LPA treatment in OAC and furthermore LPA treatment caused increased proliferation, migration and invasion in a NET1-dependent manner. NET1 knockdown resulted in reduced OAC cell proliferation and invasion.
As found in other malignancies, NET1 expression is elevated in OAC and its pre-malignant phenotype, Barrett’s oesophagus. NET1 promotes OAC cell invasion and proliferation and it mediates LPA-induced OAC cell migration.
PMCID: PMC3751529  PMID: 23945136
Neuroepithelial transforming gene 1; NET1; Oesophageal cancer; Guanine nucleotide exchange factor; Gastrointestinal oncology
14.  Synthesis and immunological properties of Vi and di-O-acetyl pectin protein conjugates with adipic acid dihydrazide as the linker. 
Infection and Immunity  1997;65(6):2088-2093.
The Vi capsular polysaccharide of Salmonella typhi, a licensed vaccine for typhoid fever in individuals > or = 5 years old, induces low and short-lived antibodies in children, and reinjection does not elicit booster responses at any age. Its immunogenicity was improved by binding Vi to proteins by using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as a linker. Similar findings were observed with the structurally related, di-O-acetyl derivative of pectin [poly-alpha(1-->4)-D-GalpA] designated OAcP. Protein conjugates of Vi and OAcP were synthesized by carbodiimide-mediated synthesis with adipic acid dihydrazide (ADH) as the linker. Hydrazide groups were introduced into proteins (bovine serum albumin or recombinant Pseudomonas aeruginosa exoprotein A) by treatment with ADH and 1-ethyl-3(3-dimethylaminopropyl carbodiimide (EDC). The resultant adipic acid hydrazide derivatives (AH-proteins), containing 2.3 to 3.4% AH, had antigenic and physicochemical properties similar to those of the native proteins. The AH-proteins were bound to Vi and OAcP by treatment with EDC. The immunogenicity of Vi or OAcP, alone or as protein conjugates, was evaluated in young outbred mice and guinea pigs by subcutaneous injection of 2.5 and 5.0 microg, respectively, of polysaccharide, and antibodies were measured by enzyme-linked immunosorbent assay. All conjugates were significantly more immunogenic than Vi or OAcP alone and induced booster responses with 5- to 25-fold increases of antibodies. Vi conjugates were significantly more immunogenic than their OAcP analogs. A carboxymethyl derivative of yeast beta-glucan enhanced the anti-Vi response elicited by an OAcP conjugate but had no effect on the immunogenicity of Vi or of OAcP alone. Vi and OAcP conjugates synthesized by this scheme will be evaluated clinically.
PMCID: PMC175288  PMID: 9169736
15.  Isolation, characterization and comparative genomics of bacteriophage SfIV: a novel serotype converting phage from Shigella flexneri 
BMC Genomics  2013;14:677.
Shigella flexneri is the major cause of shigellosis in the developing countries. The O-antigen component of the lipopolysaccharide is one of the key virulence determinants required for the pathogenesis of S. flexneri. The glucosyltransferase and/or acetyltransferase genes responsible for the modification of the O-antigen are encoded by temperate serotype converting bacteriophage present in the S. flexneri genome. Several serotype converting phages have previously been isolated and characterized, however, attempts to isolate a serotype converting phage which encodes the modification genes of serotypes 4a strain have not been successful.
In this study, a novel temperate serotype converting bacteriophage SfIV was isolated. Lysogenisation of phage SfIV converted serotype Y strain to serotype 4a. Electron microscopy indicated that SfIV belongs to Myoviridae family. The 39,758 bp genome of phage SfIV encompasses 54 open reading frames (orfs). Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. The comparative analysis also revealed that SfIV phage contained five proteins which were not found in any other phages of S. flexneri. These proteins were: a tail fiber assembly protein, two hypothetical proteins with no clear function, and two other unknown proteins which were encoded by orfs present on a moron, that presumably got introduced in SfIV genome from another species via a transposon. These unique proteins of SfIV may play a role in the pathogenesis of the host.
This study reports the isolation and complete genome sequence analysis of bacteriophage SfIV. The SfIV phage has a host range significantly different from the other phages of Shigella. Comparative genome analysis identified several proteins unique to SfIV, which may potentially be involved in the survival and pathogenesis of its host. These findings will further our understanding on the evolution of these phages, and will also facilitate studies on development of new phage vectors and therapeutic agents to control infections caused by S. flexneri.
PMCID: PMC3851460  PMID: 24090466
Shigella flexneri; Bacteriophage; O-antigen modification; Serotype conversion
16.  Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri. 
Journal of Bacteriology  1990;172(4):1905-1915.
A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.
PMCID: PMC208685  PMID: 1690703
17.  Id4 and FABP7 are preferentially expressed in cells with astrocytic features in oligodendrogliomas and oligoastrocytomas 
Oligodendroglioma (ODG) and oligoastrocytoma (OAC) are diffusely infiltrating primary brain tumors whose pathogenesis remains unclear. We previously identified a group of genes whose expression was inversely correlated with survival in a cohort of patients with glioblastoma (GBM), and some of these genes are also reportedly expressed in ODG and OAC. We examined the expression patterns and localization of these survival-associated genes in ODG and OAC in order to analyze their possible roles in the oncogenesis of these two tumor types.
We used UniGene libraries derived from GBM and ODG specimens to examine the expression levels of the transcripts for each of the 50 GBM survival-associated genes. We used immunohistochemistry and cDNA microarrays to examine expression of selected survival-associated genes and Id4, a gene believed to control the timing of oligodendrocyte development. The expression of FABP7 and Id4 and the survival of patients with ODG and OAC were also analyzed.
Transcripts of most survival-associated genes as well as Id4 were present in both GBM and ODG tumors, whereas protein expression of Id4 and one of the survival-associated genes, brain-type fatty acid-binding protein (FABP7), was present in cells with astrocytic features, including reactive and neoplastic astrocytes, but not in neoplastic oligodendrocytes. Id4 was co-expressed with FABP7 in microgemistocytes in ODG and in neoplastic astrocytes in OAC. Id4 and FABP7 expression, however, did not correlate with the clinical outcome of patients with ODG or OAC tumors.
Expression of Id4 and some of our previously identified GBM survival-associated genes is present in developing or mature oligodendrocytes. However, protein expression of Id4 and FABP7 in GBM, ODG, and OAC suggests that this group of functionally important genes might demonstrate two patterns of expression in these glioma subtypes: one group is universally expressed in glioma cells, and the other group of genes is expressed primarily in neoplastic astrocytes but not in neoplastic oligodendrocytes. Differential protein expression of these two groups of genes in ODG and OAC may be related to the cellular origins and the histological features of the neoplastic cells.
PMCID: PMC1182359  PMID: 16018821
18.  Regulation of ornithine utilization in Pseudomonas aeruginosa (PAO1) is mediated by a transcriptional regulator, OruR. 
Journal of Bacteriology  1997;179(24):7834-7842.
We have used transpositional mutagenesis of a proline auxotroph (PAO951) to isolate an ornithine utilization (oru) mutant of Pseudomonas aeruginosa (PAO951-4) that was unable to use ornithine efficiently as the sole carbon and nitrogen source. DNA sequence analysis of the inactivated locus confirmed that the transposon had inserted into a locus whose product demonstrated significant primary sequence homology to members of the AraC family of transcriptional activators. DNA mobility shift assays affirmed this potential regulatory function and indicated that the inactivated gene encodes a transcriptional regulator, which has been designated OruR. In trying to define the ornithine utilization phenotype further, a similar inactivation was engineered in the wild-type strain, PAO1. The resulting isolate (PAO1R4) was totally unable to use ornithine as the sole carbon source. Despite the intensified phenotype, this isolate failed to demonstrate significant changes in any of the catabolic or anabolic enzymes that are known to be subject to regulation by the presence of either ornithine or arginine. It did, however, show modified levels of an enzyme, ornithine acetyltransferase (OAcT), that was previously thought to have merely an anaplerotic activity. Definition of this oruR locus and its effects upon OAcT activity provide evidence that control of ornithine levels in P. aeruginosa may have a significant impact upon how the cell is able to monitor and regulate the use of arginine and glutamate as sources of either carbon or nitrogen.
PMCID: PMC179749  PMID: 9401045
19.  Mechanistic Studies on Direct Arylation of Pyridine N-Oxide: Evidence for Cooperative Catalysis between Two Distinct Palladium Centers 
Direct arylations of pyridine N-oxide (PyO), a convenient method to prepare 2-arylpyridines, catalyzed by Pd(OAc)2 and PtBu3 have been proposed to occur by the generation of a PtBu3-ligated arylpalladium acetate complex (PtBu3)Pd(Ar)(OAc) and the reaction of this complex with PyO. We provide strong evidence that (PtBu3)Pd(Ar)(OAc) does not react directly with pyridine N-oxide. Instead, our data imply that the cyclometallated complex [Pd(OAc)(tBu2PCMe2CH2)]2, which is generated from the decomposition of (PtBu3)Pd(Ar)(OAc), reacts with PyO and serves as a catalyst for the reaction of PyO with (PtBu3)Pd(Ar)(OAc). The reaction of PyO with (PtBu3)Pd(Ar)(OAc) occurs with an induction period, and the reaction of (PtBu3)Pd(Ar)(OAc) with excess PyO in the presence of [Pd(OAc)(tBu2PCMe2CH2)]2 is zero-order in (PtBu3)Pd(Ar)(OAc). Moreover, the rates of reactions of PyO with bromobenzene catalyzed by [Pd(OAc)(tBu2PCMe2CH2)]2 and [Pd(PtBu3)2] depend on the concentration of [Pd(OAc)(tBu2PCMe2CH2)]2, but not on the concentration of [Pd(PtBu3)2]. Finally, the reaction of (PtBu3)Pd(Ar)(OAc) with the model heteroarylpalladium complex containing a cyclometallated phosphine [(PEt3)Pd(2-benzothienyl)(tBu2PCMe2CH2)] rapidly formed the arylated heterocycle. Together, these data imply that the rate-determining C-H bond cleavage occurs between PyO and the cyclometallated [Pd(OAc)(tBu2PCMe2CH2)]2, rather than between PyO and (PtBu3)Pd(Ar)(OAc). In this case, the resulting heteroarylpalladium complex transfers the heteroaryl group to (PtBu3)Pd(Ar)(OAc), and C-C bondformation occurs from (PtBu3)Pd(Ar)(2-pyridyl oxide). This mechanism proposed for the direct arylation of PyO constitutes an example of C-H bond functionalization in which C-H activation occurs at one metal center, and the activated moiety undergoes functionalization after transfer to a second metal center.
PMCID: PMC3320665  PMID: 22313324
20.  Synthesis and some immunologic properties of an O-acetyl pectin [poly(1-->4)-alpha-D-GalpA]-protein conjugate as a vaccine for typhoid fever. 
Infection and Immunity  1994;62(12):5545-5549.
Pectin, a plant polysaccharide, is mostly a linear homopolymer of poly(1-->4)-alpha-D-GalpA with < 5% neutral sugars: its molecular size has a broad distribution around 400 kDa, and the degree of esterification is < 5%. The structure of the capsular polysaccharide of Salmonella typhi (Vi) differs from pectin in that it is N acetylated at C-2 and O acetylated at C-3, and has a molecular size of approximately 2 x 10(3) kDa. There is no serological cross-reaction between pectin and Vi. Pectin, when O acetylated at C-2 and C-3, is antigenically identical to Vi in double immunodiffusion. Unlike Vi, O-acetylated pectin (OAcPec) is not immunogenic in mice, probably because of its comparatively low molecular weight. After storage at 3 to 8 degrees C for 3 months, there was no change in the O-acetyl content or the M(r) of OAcPec. At 60 degrees C, the M(r) of OAcPec declined more rapidly than that of Vi. OAcPec conjugated to tetanus toxoid elicited Vi antibodies in mice, and reinjection elicited a booster response. The levels of Vi antibodies elicited by OAcPec-tetanus toxoid conjugates were lower than those elicited by Vi conjugates, but these differences were not statistically significant. OAcPec has some advantages because it can be measured by standardized colorimetric assays and because it forms more soluble conjugates with proteins than does Vi. One disadvantage is that its glycosidic bond is not as stable as that of Vi. The use of a plant polysaccharide, pectin, as an immunogen for prevention of a systemic infection caused by a capsulated pathogen (S. typhi) provides a novel approach to improve the preparation and immunogenicity of polysaccharide-based vaccines.
PMCID: PMC303300  PMID: 7960137
21.  Demographic and phenotypic features of 70 families segregating Barrett's oesophagus and oesophageal adenocarcinoma 
Journal of Medical Genetics  2003;40(9):651-656.
Background: Barrett's oesophagus (BO) also termed metaplastic columnar lined oesophageal epithelium, is believed to result from a continual reparative response to chronic reflux of gastric contents. Although traditionally considered to be an acquired disorder, with several epidemiological risk factors involved, it is now recognised that there is a genetic component, and that this is likely to be autosomal dominant. Clustering of BO and of oesophageal adenocarcinoma (OAC) or oesophagogastric junctional adenocarcinoma (OGJAC) has been shown in a number of studies.
Methods: We investigated a large series of BO/OAC families to find evidence that a subset of BO/OAC is the result of a hereditary predisposition, and explored the potential of performing a linkage study with the families identified to date.
Results: Of 957 individuals in 70 families, 173 had a reported diagnosis of BO or OAC/OGJAC: 101 had BO only, 52 had OAC/OGJAC, and 20 had both BO and OAC/OGJAC. There were 133 affected males and 40 affected females, a male:female ratio of 3.3:1. In addition, 124 participants (12.9%) had a reported diagnosis of cancer other than OAC. Of these, 15 did (affected) and 109 did not (unaffected) have a diagnosis of BO or OAC. A cancer other than OAC was found in 13.9% of unaffected and 8.7% of affected individuals.
Conclusion: It is widely accepted that the majority of BO and OAC are sporadic, although familial clustering of BO and OAC has been recognised for at least three decades. Environment and lifestyle undoubtedly play a role in development of BO and OAC, and may affect the penetrance of the putative inherited factor. Although our 70 BO probands were not more likely to develop non-OAC/OGJAC cancers than normal, over a third had developed either OAC or OGJAC, and might have gone on to develop others. We intend to continue recruitment and initiate formal linkage studies to identify the causative gene(s).
PMCID: PMC1735581  PMID: 12960209
22.  Chloride ligation in inorganic manganese model compounds relevant to Photosystem II studied using X-ray absorption spectroscopy 
Chloride ions are essential for proper function of the photosynthetic oxygen-evolving complex (OEC) of Photosystem II (PS II). Although proposed to be directly ligated to the Mn cluster of the OEC, the specific structural and mechanistic roles of chloride remain unresolved. This study utilizes X-ray absorption spectroscopy (XAS) to characterize the Mn–Cl interaction in inorganic compounds that contain structural motifs similar to those proposed for the OEC. Three sets of model compounds are examined; they possess core structures MnIV3O4X (X = Cl, F, or OH) that contain a di-μ-oxo and two mono-μ-oxo bridges or MnIV2O2X (X = Cl, F, OH, OAc) that contain a di-μ-oxo bridge. Each set of compounds is examined for changes in the XAS spectra that are attributable to the replacement of a terminal OH or F ligand, or bridging OAc ligand, by a terminal Cl ligand. The X-ray absorption near edge structure (XANES) shows changes in the spectra on replacement of OH, OAc, or F by Cl ligands that are indicative of the overall charge of the metal atom and are consistent with the electronegativity of the ligand atom. Fourier transforms (FTs) of the extended X-ray absorption fine structure (EXAFS) spectra reveal a feature that is present only in compounds where chloride is directly ligated to Mn. These FT features were simulated using various calculated Mn–X interactions (X = O, N, Cl, F), and the best fits were found when a Mn–Cl interaction at a 2.2–2.3 Å bond distance was included. There are very few high-valent Mn halide complexes that have been synthesized, and it is important to make such a comparative study of the XANES and EXAFS spectra because they have the potential for providing information about the possible presence or absence of halide ligation to the Mn cluster in PS II.
PMCID: PMC3965209  PMID: 14758524
Chloride cofactor; K-edge absorption; Manganese EXAFS; Photosystem II
23.  Molecular Structure-Affinity Relationship of Bufadienolides and Human Serum Albumin In Vitro and Molecular Docking Analysis 
PLoS ONE  2015;10(5):e0126669.
The development of bufadienolides as anti-tumor agents is limited due to poor pharmacokinetic properties regarding drug half-lives and toxicity in vivo. These serious factors might be improved by increasing the drug/albumin-binding ratio. This study therefore investigated the relationship between the structural properties of nine bufadienolides and their affinities for human serum albumin (HSA) by a fluorescence spectroscopy-based analysis and molecular docking. Fluorescence quenching data showed that the interaction of each bufadienolide with HSA formed a non-fluorescent complex, while thermodynamic parameters revealed negative ΔS and ΔH values, corresponding to changes in enthalpy and entropy, respectively. The structural differences between the various bufadienolides markedly influenced their binding affinity for HSA. With the exception of a C = O bond at the C12 position that decreased the binding affinity for HSA, other polar groups tended to increase the affinity, especially a hydroxyl (OH) group at assorted bufadienolide sites. The rank order of binding affinities for drugs with tri-hydroxyl groups was as follows: 11-OH > 5-OH > 16-OH; in addition, 16-acetoxy (OAc), 10-aldehyde and 14-epoxy constituents notably enhanced the binding affinity. Among these groups, 11-OH and 16-acetyl were especially important for a seamless interaction between the bufadienolides and HSA. Furthermore, molecular docking analysis revealed that either an 11-OH or a 16-OAc group spatially close to a five-membered lactone ring significantly facilitated the anchoring of these compounds within site I of the HSA pocket via hydrogen bonding (H-bonding) with Tyr150 or Lys199, respectively. In summary, bufadienolide structure strongly affects binding with HSA, and 11-OH or 16-OAc groups improve the drug association with key amino acid residues. This information is valuable for the prospective development of bufadienolides with improved pharmacological profiles as novel anti-tumor drugs.
PMCID: PMC4422520  PMID: 25946161
24.  Use and Associated Risks of Concomitant Aspirin Therapy with Oral Anticoagulation in Patients with Atrial Fibrillation: Insights from the ORBIT-AF Registry 
Circulation  2013;128(7):721-728.
The role of concomitant aspirin therapy in patients with atrial fibrillation (AF) receiving oral anticoagulation (OAC) is unclear. We assessed concomitant aspirin use and its association with clinical outcomes among AF patients treated with OAC.
Methods and Results
The Outcomes Registry for Better Informed Treatment (ORBIT) of Atrial Fibrillation registry enrolled 10,126 AF patients from 176 US practices from June, 2010 through August, 2011. The study population was limited to those on OAC (n=7,347).
Hierarchical multivariable logistic regression models were used to assess factors associated with concomitant aspirin therapy. Primary outcomes were 6-month bleeding, hospitalization, ischemic events, and mortality. Overall, 35% (n=2543) of AF patients on OAC also received aspirin (OAC+ASA). Patients receiving OAC+ASA were more likely male (66% vs. 53%, p<0.0001) and had more comorbid illness than those on OAC alone. Over one-third (39%) of OAC+ASA did not have a history of atherosclerotic disease, yet 17% had elevated ATRIA bleeding risk scores (≥5). Major bleeding (adjusted HR 1.53, 95% CI 1.20–1.96) and bleeding hospitalizations (adjusted HR 1.52, 95% CI 1.17–1.97) were significantly higher in those on OAC+ASA versus OAC alone. Rates of ischemic events were low.
Patients with AF receiving OAC are often treated with concomitant aspirin, even when they do not have cardiovascular disease. Use of OAC+ASA was associated with significantly increased risk for bleeding, emphasizing the need to carefully determine if and when the benefits of concomitant aspirin outweigh the risks in AF patients already on OAC.
PMCID: PMC3908483  PMID: 23861512
atrial fibrillation; anticoagulants; aspirin; hemorrhage; outcomes research
25.  Isolation and genomic characterization of SfI, a serotype-converting bacteriophage of Shigella flexneri 
BMC Microbiology  2013;13:39.
All Shigella flexneri serotypes except serotype 6 share a common O-antigen tetrasaccharide backbone and nearly all variations between serotypes are due to glucosyl and/or O-acetyl modifications of the common O unit mediated by glycosyltransferases encoded by serotype-converting bacteriophages. Several S. flexneri serotype-converting phages including SfV, SfX, Sf6 and SfII have been isolated and characterized. However, S. flexneri serotype-converting phage SfI which encodes a type I modification of serotype 1 (1a, 1b, 1c and 1d) had not yet been characterized.
The SfI phage was induced and purified from a S. flexneri serotype 1a clinical strain 019. Electron microscopy showed that the SfI phage has a hexagonal head and a long contractile tail, characteristic of the members of Myoviridae family. SfI can convert serotype Y to serotype 1a and serotype X to serotype 1d, but cannot convert 10 other S. flexneri serotypes (1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, Xv) tested, suggesting that SfI has a narrow host range. Similar to other S. flexneri serotype-converting phages, SfI integrates into the tRNA-thrW gene adjacent to proA of the host chromosome when lysogenized. The complete sequence of the SfI genome was 38,389 bp, encoding 66 open reading frames and two tRNA genes. Phage SfI shares significant homology with S. flexneri phage SfV, Escherichia coli prophage e14 and lambda, and is classified into the lambdoid phage family. SfI was found to use a cos mechanism for DNA packaging similar to that of phage SfV.
SfI contains features of lambdoid phages and is closely related to S. flexneri phage SfV, E. coli prophage e14 and lambda. The characterization of SfI enhances our understanding of serotype conversion of S. flexneri.
PMCID: PMC3636060  PMID: 23414301

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