Phase variable restriction-modification systems in Moraxella catarrhalis

A repetitive DNA motif was used as a marker to identify novel genes in the mucosal pathogen Moraxella catarrhalis. There is a high prevalence of such repetitive motifs in virulence genes that display phase variable expression. Two repeat containing loci were identified using a digoxigenin-labelled 5'-(CAAC)(6)-3' oligonucleotide probe. The repeats are located in the methylase components of two distinct type III restriction-modification (R-M) systems. We suggest that the phase variable nature of these R-M systems indicates that they have an important role in the biology of M. catarrhalis. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis. of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pgIB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pgIB2 polymorphisms were not found in strain C311#3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311#3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311#3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311#3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311#3 and other strains. We also present evidence that pglG, pglH and pgIB2 are potentially phase variable.

Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis

We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B.15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the beta-chain heptose (HepII) of the inner core. In contrast, N. neningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis.

The phasevarion: A genetic system controlling coordinated, random switching of expression of multiple genes

Several host-adapted bacterial pathogens contain methyltransferases associated with type III restriction-modification (R-M) systems that are subject to reversible, high-frequency on/off switching of expression (phase variation). To investigate the role of phase-variable expression of R-M systems, we made a mutant strain lacking the methyltransferase (mod) associated with a type III R-M system of Haemophilus influenzae and analyzed its phenotype. By microarray analysis, we identified a number of genes that were either up- or down-regulated in the mod mutant strain. This system reports the coordinated random switching of a set of genes in a bacterial pathogen and may represent a widely used mechanism.

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.

Clinicopathological significance of the 'keloid-like' collagen and myxoid stroma in advanced rectal cancer

Aim: To establish the histological categorization of fibrotic stroma which reflects the biological behaviour of advanced rectal cancer. Methods and results: Six hundred and twenty-seven surgically resected cases of advanced rectal carcinoma were examined. We histologically categorized fibrotic stroma in the invasive frontal region into three groups: type A, multiple fine and mature fibres were stratified into layers: type B, broad bands of eosinophilic hyalinized collagen ('keloid-like' collagen) were intermingled: type C, myxoid stroma. Type A stroma was observed in 63% of patients, type B stroma in 25%, type C stroma in 12%.. The incidence of type A stroma decreased in accordance with Dukes stage (98% in Dukes A: 73% in B: 41%, in C1: 29% in C2) and conversely, there was an increase of C type (0%, in Dukes A; 4%, in B: 20% in C1: 54% in C2). Stroma type had a significant correlation with long-term survival (80% of 5-year survival in type A stroma: 54% in type B: 26% in type C). Based on multivariate analysis. it was found that the stromal pattern had independent prognostic value, together with nodal involvement. growth pattern. and lymphocyte infiltration. Conclusions: Tumour fibrotic stroma may play an important role as a regulator of neoplastic behaviour. Pathological categorization of the fibrotic stroma is helpful for predicting the prognostic outcome of patients with rectal carcinoma.

The therapeutic potential of endothelin-1 receptor antagonists and endothelin-converting enzyme inhibitors on the cardiovascular system

Clinical trials have established bosentan, an orally active non-selective endothelin (ET) receptor antagonist, as a beneficial treatment in pulmonary hypertension. Trials have also shown short-term benefits of bosentan in systemic hypertension and congestive heart failure. However, bosentan also increased plasma levels of ET-1, probably by inhibiting the clearance of ET-1 by endothelin type B (ET.) receptors, and this may mean its effectiveness is reduced with long-term clinical use. Preliminary data suggests that selective endothelin type A (ETA) receptor antagonists (BQ-123, sitaxsentan) may be more beneficial than the non-selective ET receptor antagonists in heart failure, especially when the failure is associated with pulmonary hypertension. Experimental evidence in animal disease models suggests that non-selective ET or selective ETA receptor antagonism may have a role in the treatment of athero-sclerosis, restenosis, myocarditis, shock and portal hypertension. In animal models of myocardial infarction and/or reperfusion injury, non-selective ET or selective ETA receptor antagonists have beneficial or detrimental effects depending on the conditions and agents used. Thus clinical trials of the nonselective ET or selective ETA receptor antagonists in these conditions are not presently warranted. Several selective endothelin-converting enzyme inhibitors tors have been synthesised recently, and these are only beginning to be tested in animal models of cardiovascular disease, and thus the clinical potential of these inhibitors is still to be defined.

The lateral line canal sensory organs of the Epaulette Shark (Hemiscyllium ocellatum)

Examination of the lateral line canals in the Epaulette Shark reveals a much more differentiated sensory system than previously reported from any elasmobranch. Two main types of lateral line canals are found. In one type rounded patches of sensory epithelia are separated by elevations of the canal floor. The other type is a straight canal without restrictions and with an almost continuous sensory epithelium. In addition, we found epithelia (type A) with very long apical microvilli on the supporting cells. These microvilli reach beyond the stereovilli of the hair cells. Another type (B) of sensory epithelium has short microvilli on the supporting cells. In this latter type of epithelium the stereovilli of the hair cells are comparatively tall and reach out beyond the supporting cell microvilli. New hair cells are found widely in both types of sensory epithelia. These always occur as single cells, unlike those described in teleost lateral line canal sensory epithelia where new hair cells seem to form in pairs. Dying hair cells are also widespread, indicating a continuous turnover of hair cells.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

Similar antibody concentrations in Filipino infants at age 9 months, after 1 or 3 doses of an adjuvanted, 11-valent pneumoccoccal diphtheria/tetanus-Conjugated Vaccine: a randomized controlled trial

In Filipino infants, 1 dose of an adjuvanted, 11-valent pneumococcal conjugate vaccine (serotypes 1, 4, 5, 7F, 9V, 19F, and 23F conjugated to tetanus protein; and serotypes 3, 6B, 14, and 18C conjugated to diphtheria toxoid) administered alone at age 18 weeks (11PncTD1) elicited similar antibody concentrations at age 9 months as those elicited by 3 doses (11PncTD3) administered concomitantly with national program vaccines, at ages 6, 10, and 14 weeks. Geometric mean antibody concentrations ranged from 0.36 mug/mL ( for serotype 18C) to 5.81 mug/mL (for serotype 4), for the 11PncTD1 vaccine, and from 0.32 mug/mL (for serotype 18C) to 5.01 mug/mL (for serotype 19F), for the 11PncTD3 vaccine. The proportion of infants with threshold antibody concentrations greater than or equal to0.35 mug/mL was also similar (ranges, 55.6%-100% for the 11PncTD1 vaccine and 42.9%-100% for the 11PncTD3 vaccine). The functional activity of antibodies expressed as opsonophagocytic activity titers was similar in the 11PncTD1 and 11PncTD3 groups. This finding is an important one for countries with financial constraints and high pneumococcal disease burden.

Cross-reactivity of GroEL antibodies with human heat shock protein 60 and quantification of pathogens in atherosclerosis

Background/aims: Chronic infections such as those caused by Chlamydia pneumoniae and periodontopathic bacteria such as Porphyromonas gingivalis have been associated with atherosclerosis, possibly due to cross-reactivity of the immune response to bacterial GroEL with human heat shock protein (hHSP) 60. Methods: We examined the cross-reactivity of anti-GroEL and anti-P. gingivalis antibodies with hHSP60 in atherosclerosis patients and quantified a panel of six pathogens in atheromas. Results: After absorption of plasma samples with hHSP60, there were variable reductions in the levels of anti-GroEL and anti-P. gingivalis antibodies, suggesting that these antibodies cross-reacted with hHSP60. All of the artery specimens were positive for P. gingivalis. Fusobacterium nucleatum, Tannerella forsythia, C. pneumoniae, Helicobacter pylori, and Haemophilus influenzae were found in 84%, 48%, 28%, 4%, and 4% of arteries, respectively. The prevalence of the three periodontopathic microorganisms, P. gingivalis, F. nucleatum and T. forsythia, was significantly higher than that of the remaining three microorganisms. Conclusions: These results support the hypothesis that in some patients, cross-reactivity of the immune response to bacterial HSPs including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may be a possible mechanism for the association between atherosclerosis and periodontal infection.

The five families of sucrose-phosphate synthase genes in Saccharum spp. are differentially expressed in leaves and stem

Sucrose-phosphate synthase (SPS) is a key enzyme in the pathway of sucrose synthesis. Five different gene families encoding SPS have been reported in the Poaceae [Castleden CK, Aoki N, Gillespie VJ, MacRae EA, Quick WP, Buchner P, Foyer CH, Furbank RT, Lunn JE (2004) Evolution and function of the sucrose-phosphate synthase gene families in wheat and othergrasses. PlantPhysiology 135, 1753-1764]. Expression of the five families in leaf and stem tissues of Saccharum spp. at different stages of development was determined by quantitative real-time PCR. The type B and C families of SPS genes were predominantly expressed in both immature and mature leaves, whereas the two subfamilies making up the type D family were expressed at similar levels in all tissues examined. In the type A family, expression was lowest in leaves and increased from the meristem region down to internode 7 of the stem.

Thermal evolution of the ore-hosting Isa Superbasin: Central and Northern Lawn Hill Platform

Hydrocarbon migration pathways and organic mineral matter associations were used to identify brine pathways in Paleoproterozic to early Mesoproterozoic rocks from the Lawn Hill platform, Mount Isa. Several types of organic matter are identified, and their thermal imprints are used to reconstruct the thermal history of the northern to central parts of the Isa superbasin. Three major thermal hydrothermal episodes are recognized from the organic maturation studies. Isotherm plots on a 175-km-long structural-sedimentological north-south section of the Isa superbasin highlight specific fault systems that acted as hot fluid conduits during the geologic history of the basin. Some of these systems indicate continuing activity into the south Nicholson basin, supported by the presence of low reflectance (type B) bitumen. This bitumen has not been overprinted by later hydrothermal episodes and therefore represents the latest thermal event. Along the north-south profile a general southward increase in temperature is evident. The lowest temperatures are recorded in proximity to the basin margin on the southern flank of the Murphy inlier. Thermal processes and their sequence of events in the basin are recorded by organic maturation, subsequent hydrocarbon generation, its migration and destruction coincident with transport and precipitation of minerals. As some timing and trapping mechanisms for minerals may have analogues with hydrocarbon entrapment, relative timing of processes leading to organic maturation, hydrocarbon generation and migration are utilized in this study to enhance understanding of ore-grade mineralization. In the Proterozoic successions of the Mount Isa basin multiple hydrocarbon generation events are recognized. These events record the transient passage of potential metal-bearing fluids rather than background conductive heat flow from the basement. Such hydrothermal fluids are responsible for inverse maturation profiles in the vicinity of the Termite Range fault and extreme maturation (reflectance values) up to 6 percent Ro at the Grevillea prospect. At Century, intermediate Ro values of

Unicellular pheromone glands of the pentatomid bug Nezara viridula (Heteroptera : Insecta): Ultrastructure, classification, and proposed function

Male Nezara viridula produce sex pheromones from many independent single cells, each with a duct that opens onto the ventral abdominal surface. Despite the presence of along duct and an associated end complex (in the form of a cupule and microvillus saccule), the structural organization of the cells that comprise the gland conform to Class 1 epidermal gland cell classification : a single cell surrounds the entire secretory complex. Each cuticular cupule contains a central bed of filaments and opens into a narrow tubular ductule that leads from the base of the cupule through the epidermis to the cuticle to open externally as a pore. The cuticle of the cupule is continuous with that of the ductule and has the appearance of three layers, although the inner (middle) layer may be a gap formed during construction of the complex. In young adult males, just molted, the ultrastructure of the cells and their inclusions indicate that they are not active. The region of the cell that is distal to the abdominal cuticle is reduced and the proximal region, surrounding the duct, is enlarged when compared with sexually mature (3-4 weeks old) adult males. At maturity the pheromone cells are enlarged distally around the cupule, but are reduced to a narrow sleeve proximally, around the ductule. Two characteristic cell profiles are evident, based on the shape of the cupule and the organelle content. Type A shows a broad opening to the cupule, an abundance of mitochondria, and few vesicular bodies. Type B has an elongated, narrow, vase-like opening to the cupule, few mitochondria, and numerous vesicular bodies. Type B cells are smaller and more abundant than Type A. Distribution within the epidermal layer also differs. It is likely that the different types represent cells producing different secretion profiles. However, the secretions retained by the standard fixation protocol within mature cells of both types look similar and appear to collect as crystalline bodies within the lumen. This may represent a common storage mechanism.