Hussein Masoud

Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis

The availability of the complete 1.83‐megabase‐pair sequence of the Haemophilus influenzae strain Rd genome has facilitated significant progress in investigating the biology of H. influenzae lipopolysaccharide (LPS), a major virulence determinant of this human pathogen. By searching the H. influenzae genomic database, with sequences of known LPS biosynthetic genes from other organisms, we identified and then cloned 25 candidate LPS genes. Construction of mutant strains and characterization of the LPS by reactivity with monoclonal antibodies, PAGE fractionation patterns and electrospray mass spectrometry comparative analysis have confirmed a potential role in LPS biosynthesis for the majority of these candidate genes. Virulence studies in the infant rat have allowed us to estimate the minimal LPS structure required for intravascular dissemination. This study is one of the first to demonstrate the rapidity, economy and completeness with which novel biological information can be accessed once the complete genome sequence of an organism is available.

The Structure of the Lipid A Component of Rhodocyclus gelatinosus Dr2 Lipopolysaccharide

Summary Differences in lipid A and lipopolysaccharide (LPS) composition were observed between strains of sero-/ chemotype I and sero-/chemotype II of the phototrophic species Rhodocyclus gelatinosus . The lipopolysaccharides only from representative strains of sero-/chemotype I (e. g. 29/1) contained ethanolamine and ethanolamine-phosphate. In contrast, galacturonic acid was present only in strains of sero-/chemotype II. The isolated lipid A of R. gelatinosus Dr 2 , characterized by LD-MS and 31 P-NMR, showed a composition similar to that of strain 29/1 but, in contrast, lacked ethanolamine. It is constituted of a β-1,6-glucosamine disaccharide substituted at 1 and 4′-positions by phosphomonoesters. The amino groups of the disaccharide were acylated by 3-hydroxycapric acid (3-OH-10 : 0) and these in turn were substituted by lauric or myristic acids. Hydroxyl groups at C-3 and C-3′ were esterified also by 3-hydroxycapric acid. Lipid A of R. gelatinosus strains from both sero-/chemotypes showed a high cross-reactivity with lipid A of Salmonella , both in passive hemolysis and in the hemolysis inhibition test. The ratio unsaturated/saturated fatty acids of lipid A varied depending on the growth temperature (30, 20, or 14°C). The relative amounts of unsaturated fatty acids increased at the expense of saturated fatty acids when the bacteria were grown at lower temperatures. No changes were observed with the other LPS components.

The structure of the lipid A component of Sphaerotilus natans

The lipopolysaccharide of Sphaerotilus natans afforded a ladder-like pattern of bands in sodium deoxycholate-polyacrylamide gel electrophoresis, indicating the presence of a S-form lipopolysaccharide. The chemical analysis showed neutral sugars (rhamnose, glucose, l-glycero-d-manno-heptose), 3-deoxy-octulosonic acid (Kdo), amino compounds (glucosamine, glucosamine phosphate, ethanolamine and ethanolamine phosphate), and phosphorus. The lipid A fraction contained saturated and unsaturated capric, lauric, and myristic acids, and 3-hydroxy capric acid (3-OH-10:0). Its chemical structure was consisting of a glucosamine disaccharide, glycosidically substituted by a phosphomonoester, and substituted at C-4′ by a pyrophosphodiester esterified with ethanolamine. The amino groups of both glucosamines are acylated by 3-hydroxy capric acids and these in turn are substituted by saturated and unsaturated capric, lauric, and myristic acids. Hydroxyl groups of the backbone disaccharide at C-3 and C-3′ were also esterified by 3-hydroxy capric acid, those at C-4 and C-6 were unsubstituted. The latter provides the attachment site for Kdo.

A unique glycosyltransferase involved in the initial assembly of Moraxella catarrhalis lipooligosaccharides

Moraxella catarrhalis express three predominant forms of lipooligosaccharide (LOS) molecules on the bacterial surface. These major glycolipids contain specific carbohydrate epitopes that distinguish each glycoform into serotype A, B, or C LOS. All three serotypes, however, share a common glucose containing inner-core structure, consisting of an alpha-glucose attached to 2-keto-3-deoxyoctulosonic acid (KDO), which is unique among Gram-negative bacteria. Many of the LOS glycosyltransferase genes (lgt) responsible for assembly of the extended M. catarrhalis LOS structure have been identified. In this report, we now describe the identification and characterization of Lgt6, a unique glycosyltransferase that is responsible for the addition of the first glucose to the inner core thus initiating the assembly of full length LOS. Isogenic mutants defective in the expression of lgt6 were constructed in all three M. catarrhalis LOS serotypes and the resulting LOS glycoforms consisted of KDO(2)-lipid A-OH as analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. In addition, the expression of lgt6 in trans in a heptose-deficient Neisseria meningitidis NMB gmhX mutant resulted in the addition of a hexose to the LOS of this strain. These studies demonstrate that Lgt6 functions as an alpha-(1-5)-glucosyltransferase in M. catarrhalis adding the primary glucose to the KDO(2)-lipid A-OH in LOS biosynthesis. The function of Lgt6 is required for the completion of both the major and minor oligosaccharide chains in M. catarrhalis.

LPS-based conjugate vaccines composed of saccharide antigens of smooth-type Salmonella enteritidis and rough-type S. gallinarum 9R bound to bovine serum albumin

Lipopolysaccahrides (LPSs) from Salmonella enteritidis, S. gallinarum, and S. enterica Typhimurium showed an identical electrophoretic banding pattern and serological cross-reactions among each other. LPSs from wild-type Salmonella enteritidis and rough mutant S. gallinarum 9R were detoxified by cleavage of lipid A moieties using mild acid hydrolysis. The non-toxic saccharide moieties from both strains were coupled directly to bovine serum albumin (BSA). The conjugates were injected in mice in combination with monophosphory lipid A (MPL), Freund, and Alum adjuvants. The highest IgM and IgG titres were obtained when the conjugates were emulsified with MPL adjuvant, followed by Freund adjuvant. The antisera raised against the conjugates in combination with MPL and Freund adjuvants showed high complement-mediated lysis to the homologous strains. A correlation was observed between IgG titres and bactericidal activities against homologous strains. Low in vivo protection was obtained when mice immunized with the conjugates were challenged with 10 times the LD50 of the wild S. enteritidis.

Use of Moraxella catarrhalis Lipooligosaccharide Mutants To Identify Specific Oligosaccharide Epitopes Recognized by Human Serum Antibodies

ABSTRACT Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.

Structural elucidation of the specific capsular polysaccharide of Rhodococcus equi serotype 7

Structural analysis of the specific capsular polysaccharide produced by Rhodococcus equi serotype 7 indicated it to be a high-molecular-weight polymer consisting of equal molar amounts of D-galactose, D-mannose, L-rhamnose, and pyruvic acid. By employing a combination of chemical and NMR techniques, it was established that the polysaccharide is composed of the linear repeating trisaccharide units: [formula: see text] -->3)-alpha-D-Galp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-L-Rhap -(1-->, in which the cyclic pyruvic acid acetal groups bridging the O-4 and O-6 positions of the alpha-D-Manp residues have the S-configuration. The 1H and 13C NMR spectra of the native and pyruvic acetal-free polysaccharides were fully assigned.

Common Antimicrobial Resistance Patterns, Biotypes and Serotypes Found among Pseudomonas aeruginosa Isolates from Patient's Stools and Drinking Water Sources in Jordan

Abstract Pseudomonas aeruginosa was isolated in low rates from stool specimens of outpatients and inpatients (7% versus 12%) but in higher rates from chlorinated and nonchlorinated water sources (15% versus 44%), respectively in Jordan. The same biotype was recognized among 90% of P. aeruginosa isolates from patients stools and water sources using specific biochemical profiles. Three serogroups belonging to 01, 06 and 011 accounted for the majority of these isolates in water (66%) and stools (78%), respectively. All P. aeruginosa isolates from water were highly susceptible (87%-100%) to piperacillin-tazobactam, amikacin, gentamicin, imipenem, aztreonam, ceftazidime and ciprofloxacin, whereas the isolates from stool were slightly less susceptible (81%-98%) to these antimicrobials. P. aeruginosa isolates from water and stool sources were almost equally highly resistant to tetracycline (86%-89%) and carbenicillin (88%-89%), respectively. One common small plasmid (15.4 kb) was detected in 14/25 (56%) of multidrug-resistant P. aeruginosa isolates from both water and stool. This study demonstrates certain common epidemiological characteristics including antimicrobial resistance pattern, biotypes and serotypes among P. aeruginosa isolates from patients stools and drinking water sources in Jordan.

The structure of the core region of the lipopolysaccharide from Rhodocyclus gelatinosus Dr2

Summary The core oligosaccharide of Rhodocyclus gelatinosus Dr2 was isolated and its chemical structure examined by methylation analysis. The anomeric configurations of the linkages were determined by 1H-and 13C-nuclear magnetic resonance spectroscopy. The core contained one residue of 3-deoxy-D-mannooctulosonic acid (Kdo), which was assumed to be ketosidically linked to C-6′ of the non-reducing glucosamine of lipid A. The Kdo unit was substituted at C-4 by an α-1′4-linked galactopyranosuronic acid disaccharide and at C-5 by D-glycero-D-manno-heptopyranose, the latter of which was partially substituted by phosphate at C-7.

Structural elucidation of the novel core oligosaccharide from LPS of Burkholderia cepacia serogroup O4

Lipopolysaccharide (LPS) is an important virulence factor of Burkholderia cepacia, an opportunistic bacterial pathogen that causes life-threatening disease in cystic fibrosis patients and immunocompromised individuals. B. cepacia LPS comprises an O-specific polysaccharide covalently linked to a core oligosaccharide (OS) which in turn is attached to a lipid A moiety. The complete structure of the LPS core oligosaccharide from B. cepacia serotype O4 was investigated by detailed NMR and mass spectrometry (MS) methods. High- (HMW) and low-molecular-weight (LMW) OSs were obtained by deacylation, dephosphorylation, and reducing-end reduction of the LPS. Glycan and NMR analyses established that both OSs contain a common inner-core structure consisting of D-glucose, L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, 3-deoxy-D-manno-octulsonic acid, and D-glycero-D-talo-2-octulosonic acid. The structure of the LMW OS differed from that of the HMW OS in that it lacks a tetra-rhamnosyl GlcNAc OS extension. These structural conclusions were confirmed by tandem MS analyses of the two OS fractions as well as an OS fraction obtained by alkaline deacylation of the LPS. The location of a phosphoethanolamine substituent in the core region was determined by ESI-MS and methylation analysis of O-deacylated LPS and core OS samples. A polyclonal antibody to B. cepacia serotype O4 core OS was cross-reactive with several other serotypes indicating common structural features.