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PubMed2000

Antigenic diversity of Haemophilus somnus lipooligosaccharide: phase-variable accessibility of the phosphorylcholine epitope.

The lipooligosaccharide (LOS) of Haemophilus somnus undergoes antigenic phase variation, which may facilitate evasion from the bovine host immune response and/or colonization and dissemination. However, LOS antigenic diversity in H. somnus has not been adequately investigated. In this study, monoclonal antibodies (MAbs) specific to various LOS epitopes were used to investigate antigenic variation and stability in LOS from H. somnus strains and phase variants. Clinical isolates of H. somnus exhibited intrastrain, as well as interstrain, antigenic heterogeneity in LOS when probed with MAbs to outer core oligosaccharide epitopes in an enzyme-linked immunosorbent assay (ELISA). However, epitopes reactive with MAbs directed predominately to the inner core heptose region were highly conserved. At least one epitope, which was expressed in few strains, was identified. One LOS component affected by phase variation was identified as phosphorylcholine (PCho), which is linked to the primary glucose residue. Inhibition ELISA, immunoblotting, and electrospray-mass spectrometry were used to confirm that MAb 5F5.9 recognized PCho. LOS reactivity with MAb 5F5.9 was associated with loss of most of the outer core oligosaccharide, indicating that reactivity with PCho was affected by phase variation of the glucose residues in this region. Our results indicate that outer core epitopes of H. somnus LOS exhibit a high degree of random, phase-variable antigenic heterogeneity and that such heterogeneity must be considered in the design of vaccines and diagnostic tests.

Animals↗
PubMed2002

Mutator clones of Neisseria meningitidis in epidemic serogroup A disease.

Serogroup A Neisseria meningitidis has repeatedly caused widespread epidemics of meningitis and septicemia throughout the 20th century. Recently, in a limited collection of strains, epidemic serogroup A isolates were found to have elevated mutation rates that was caused by defects in mismatch repair pathways. To ascertain the role of these mutators in the epidemic spread of this serogroup, the prevalence of hypermutability in a collection of 95 serogroup A N. meningitidis invasive isolates was determined. Overall mutability in Neisseriae can be described by measuring both missense mutation rates as well as phase variation frequencies of "contingency loci." Fifty-seven percent of serogroup A isolates possessed elevated mutability, which could be divided into two classes: intermediate and high level. Eleven of 20 high-level mutators, with phase variation rates >100-fold higher than wild-type isolates, were defective in mismatch repair. Ten of the 34 intermediate mutators possessing >10-fold increases in phase variation rates could be partially complemented by a wild-type mutL allele. A high prevalence of mutators in epidemic isolates indicates that hypermutability may play a major role in the transmission of this pathogen. The added diversity derived from increased phase variation rates may allow fixation of mutator alleles more frequently during epidemic spread.

Alleles↗
PubMed2004

[Determination of partition coefficient of dissolved gases in transformer oil using phase ratio variation method and static headspace gas chromatography].

The partition coefficients of dissolved gases in transformer oil were determined using a phase ratio variation method and static headspace gas chromatography (GC). A pressure balancing and gas volume-metering device was connected to the vent of a sample loop on a six-port injection valve of the GC. The gas phase sample from the headspace vial of 25 mL was transferred to an 80 microL sample-loop through a fused silica capillary of 0.53 mm i.d., and then separated and determined quantitatively by GC. A 2 m x 1 mm i.d. GDX502 micro-packed column was used for the separation. Five different gas-liquid volume ratios in the headspace vials were measured at different equilibrium concentrations. The partition coefficients of hydrocarbon gases including methane, acetylene, ethylene, ethane and propane dissolved in transformer oil were determined by using linear regression analysis at 20 degrees C and 50 degrees C separately. The errors between the real values and regression values from experimental data were less than 4.14% except methane. Fundamental data for on-line measurement of dissolved gases in transformer oil are provided by GC.

Acetylene↗
PubMed1995

Phenotypic variation in Haemophilus influenzae: the interrelationship of colony opacity, capsule and lipopolysaccharide.

H. influenzae type b strains show phase variation between opaque (O) and translucent (T) colony phenotypes. These phenotypic differences have been related to differences in virulence for infant rats. This study shows that the switch between O and T colony phenotypes is associated with variation in the amount of cell-associated capsule in the serotype b strains Rd:b+:01, RM7004 and Eagan. O colonies comprised organisms which were more serum resistant and had more cell-associated polyribosyl ribitol phosphate (PRP) than organisms from T colonies. Strain Rd, the non-encapsulated parent of the encapsulated transformant Rd:b+:01, was constitutively translucent, consistent with its lack of capsule expression. Since previous studies had correlated O-T switching with differences in the relative molecular weight of lipopolysaccharide (LPS), LPS phenotypes of Rd and Rd:b+:01 were compared and correlated with opacity phenotype at the individual colony level. Both strains showed phase variation between higher and lower molecular weight LPS oligosaccharide structures but the prevalence of higher molecular weight LPS was greater for the capsule-deficient Rd than encapsulated Rd:b+01. Capsule-deficient mutants of strains Rd:b+:01, RM7004 and Eagan produced constitutively translucent colonies and each had a greater prevalence of higher molecular weight LPS than their encapsulated parents. These findings indicated an incomplete association between capsular O-T phase variation and LPS expression.

Blood Bactericidal Activity↗
PubMed1997

Transcriptional analysis of mga, a regulatory gene in Streptococcus pyogenes: identification of monocistronic and bicistronic transcripts that phase vary.

Transcription of several surface virulence proteins of Streptococcus pyogenes is regulated by Mga, a protein that shows homology to response regulators of two-component signal-transducing systems. Two of these surface virulence proteins, M protein and C5a peptidase, undergo phase variation. To determine whether Mga itself undergoes phase variation and might allow the phasing switch to coordinate the activity of these genes, expression of the mga gene was analyzed. We show for the first time that there are two mga-specific transcripts: a 3.8-kb bicistronic message that includes both mga and emm12 genes and a monocistronic 1.6-kb mga message. Both transcripts phase vary and are present in higher amounts in M+ variants than in M- variants. Incubation of RNA with rifampicin indicates that the smaller 1.6-kb message is not a processed product. Two promoters were mapped upstream of mga: P1 at position 666 (-395) and P2 at position 978 (-83). In strain CS46 (delta mga), transcription initiation from the P1 promoter does not occur, and multiple start sites are found around the P2 promoter. Complementation experiments indicate that sequences upstream of the P2 promoter are required for activation of emm12 and scpA by Mga in trans.

Amino Acid Sequence↗
PubMed1993

Cell killing and chromosomal aberrations induced in Chinese hamster ovary cells by treating with cisplatin at 41.5 degrees C during the G1 or late S phase.

Variation in sensitivity to cisplatin during the cell cycle was studied in synchronous Chinese hamster ovary cells treated during G1 or late S for 1 h at 41.5 degrees C with cisplatin (0.25-1.25 micrograms/ml, 0.8-4.2 x 10(-6) M). The cells were assayed for cell killing and chromosomal aberrations. Either they were plated for colony survival, or colcemid was added from 12 to 40 h after plating followed by fixation 4 h later for analysis of chromosomal aberrations after the cells completed 1 or 2 cycles (i.e., first or second mitosis). When the cells were treated either in G1 or late S, the cells entering metaphase exhibited primarily chromatid-type deletions and exchanges. However, aberrations were observed primarily in the first mitosis when cells were treated in G1 compared with aberrations being observed in both the first and second mitoses when cells were treated in late S. For a given amount of cytotoxicity or cytological damage, the cisplatin concentration at 41.5 degrees C could be reduced 4-6-fold compared with treatment at 37 degrees C. For low cisplatin concentrations of less than 0.5-0.7 microgram/ml (survival, approximately 0.3), heat killing predominated, and cells treated in S phase were more sensitive than those treated in G1. However, for cisplatin concentrations greater than 0.5-0.7 microgram/ml, cisplatin cytotoxicity predominated, and for both cell killing and chromosomal aberrations, the cells treated in G1 were approximately 1.5 times more sensitive than those treated in late S. Furthermore, the positive correlation between survival and aberration frequency was similar for cells treated at 37 degrees C or 41.5 degrees C in either G1 or late S. These results suggest that cisplatin administered at 37 degrees C or 41.5 degrees C causes cell lethality primarily by the induction of chromosomal aberrations.

Animals↗
PubMed1986

Analysis of cell-growth-phase-related variations in hyaluronate synthase activity of isolated plasma-membrane fractions of cultured human skin fibroblasts.

Hyaluronate synthase activity is localized exclusively in plasma-membrane fractions of cultured human skin fibroblasts. The enzyme activity of plasma membranes prepared from exponential-growth-phase cells was about 6.5 times that of stationary-growth-phase cells. Hyaluronate synthase from exponential-growth-phase cells exhibited lower Km and higher Vmax. values for both UDP-N-acetylglucosamine and UDP-glucuronic acid and higher rate of elongation of hyaluronate chains compared with the enzyme from stationary-growth-phase cells. Hyaluronate synthase exhibited an extremely short half-life, 2.2 h and 3.8 h respectively when cells were treated with cycloheximide and actinomycin D. The cell-growth-phase-dependent variations in hyaluronate synthase activity appear to be due to its high turnover rate as well as due to some post-translational modification of the enzyme protein as cells progress from early exponential to stationary growth phase. The isolated plasma membranes contained a protein (Mr approx. 450,000) that was selectively autophosphorylated from [gamma-32P]ATP in vitro in the presence of hyaluronate precursors in the reaction mixture and that also exhibited some hyaluronate-synthesis-related properties. The 32P-labelled protein isolated from plasma membranes of exponentially growing cells expressed an efficient UDP-[14C]glucuronic acid- and UDP-N-acetyl[3H]glucosamine-binding activity and was able to synthesize oligosaccharides (Mr 5000) of [14C]glucuronic acid and N-acetyl[3H]glucosamine residues. The corresponding protein of stationary-growth-phase cells, which expressed much higher nucleotide-sugar-precursor-binding activity, appeared to have lost its oligosaccharide-synthesizing activity.

Adenosine Triphosphate↗
PubMed1991

Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5' regulatory sequences.

Antigenic diversity is generated in the wall-less pathogen Mycoplasma hyorhinis by combinatorial expression and phase variation of multiple, size-variant membrane surface lipoproteins (Vlps). The unusual structural basis for Vlp variation was revealed in a cluster of related but divergent vlp genes, vlpA, vlpB and vlpC, which occur as single chromosomal copies. These encode conserved N-terminal domains for membrane insertion and lipoprotein processing, but divergent external domains undergoing size variation by loss or gain of repetitive intragenic coding sequences while retaining a motif with distinctive charge distribution. Genetic analysis of phenotypically switched isogenic lineages representing ON or OFF expression states of Vlp products ruled out chromosomal rearrangement or frameshift mutations as mechanisms for Vlp phase variation. However, highly conserved vlp promoter regions contain a tract of contiguous A residues immediately upstream of the -10 box which is subject to frequent mutations altering its length in exact correspondence with the ON and OFF phase states of specific genes. This suggests a mechanism of transcriptional control regulating high frequency phase variation and random combinatorial expression of Vlps. The multiple levels of diversity embodied in the vlp gene cluster represents a novel adaptive capability particularly suited for this class of wall-less microbe.

Amino Acid Sequence↗
PubMed1995

Variable expression of class 1 outer membrane protein in Neisseria meningitidis is caused by variation in the spacing between the -10 and -35 regions of the promoter.

The class 1 outer membrane protein encoded by the porA gene of Neisseria meningitidis is a candidate for a vaccine against meningococcal infection. The expression of class 1 outer membrane protein displays phase variation between three expression levels. Northern (RNA) blot and primer extension analysis revealed that this phase variation is regulated at the transcriptional level. The start site for transcription is located 59 bp upstream of the translational initiation codon. Sequence analysis of the promoter region of the porA gene of a variant without class 1 protein expression revealed nine contiguous guanidine residues between the -10 and -35 domains. Comparison of promoter sequences of different phase variants indicated that the length of the polyguanidine stretch correlated with the expression level of the class 1 outer membrane protein; the presence of 11, 10, or 9 contiguous guanidine residues results in high levels, medium levels, or no expression of class 1 mRNA, respectively. These results suggest that the variable porA expression levels seen in different isolates are modulated by guanidine residue insertion and/or deletion due to slipped-strand mispairing on the polyguanidine stretch within the intervening sequence of the -35 and -10 regions of the promoter. The phase variation of class 1 outer membrane protein may provide a molecular mechanism to evade the host immune defense. Therefore, the protective efficacy of a vaccine based on class 1 outer membrane protein may be questioned.

Bacterial Vaccines↗
PubMed1993

Determination of colchicine in biological fluids by reverse-phase HPLC. Variation of colchicine levels in rats.

A reverse-phase HPLC method for the determination of colchicine in biological fluids is proposed. Blood, liver and kidney colchicine concentrations were determined in rats at different times following intraperitoneal (i.p.) injection of 10 mg/kg of the drug. Colchicine was extracted from the samples studied using dichloromethane at pH 8. The dry extract was redissolved in the mobile phase and analyzed with simultaneous detection at 254 and 350 nm.

Animals↗
PubMed1993

Variation and genetic control of surface antigen expression in mycoplasmas: the Vlp system of Mycoplasma hyorhinis.

Surface antigenic diversity in the swine pathogen Mycoplasma hyorhinis is generated by random combinatorial expression and high-frequency phase variation of multiple, size-variant membrane surface lipoproteins (Vlps) which represent the major coat proteins of this wall-less procaryote. The distinctive structural basis for Vlp variation was revealed in a family of several related but divergent vlp genes. These occur in one cluster as single chromosomal copies, each encoding a conserved domain for membrane insertion and lipoprotein processing, and a divergent external domain that changes size by deletion or insertion of repetitive intragenic coding sequences while retaining a distinctive charge motif. Lack of detectable changes in restriction fragment patterns or DNA sequence of vlp structural genes during phase transitions between ON and OFF expression states ruled out long range genomic rearrangements and frameshift mutations as a means of controlling Vlp phase variation. However, highly homologous vlp promoter regions contain a homopolymeric tract of contiguous adenine residues [poly(A)] upstream of the transcriptional start site which is subject to frequent mutations altering its length. These mutations are the only sequence changes detected during phase transitions, and are highly correlated with the expression state of each vlp gene. This suggests a mechanism of transcriptional control regulating Vlp phase variation by critical changes within the poly(A) region affecting the spacing between the -10 and -35 hexamers or a putative regulator binding site. The multiple levels of structural and antigenic diversity embodied in the vlp gene family may provide essential adaptive capabilities for this wall-less microbial pathogen.

Animals↗
PubMed1989

Production of the fimbrial adhesin 987P by enterotoxigenic Escherichia coli during growth under controlled conditions in a chemostat.

The effects of growth conditions on the production of 987P fimbriae by the enterotoxigenic Escherichia coli strain 1592 were examined in steady state chemostat experiments at different specific growth rates. The amount of fimbriae produced by fimbriate cells (P+) was dependent on the specific growth rate (mu). Under aerobic growth conditions fimbriae production increased with higher mu values till mu = 0.40 h-1 and decreased again at mu values close to mu max (0.48 h-1). Under anaerobic growth conditions the maximal production was comparable to that under aerobic growth conditions, and was also maximal close to mu max (0.16 h-1). Phase variation, measured as the percentage of fimbriate cells in a particular population, was independent of mu. The composition of the growth medium influenced both phase variation and overall production of fimbriae. A shift from minimal to a complex medium induced a rapid reduction in the amount of fimbriae per P+ cell and a slower reduction in the percentage of P+ cells. A shift from complex to minimal medium resulted in an increase in the percentage of P+ cells and a constant amount of fimbriae per P+ cell. The frequency of the phase switch was calculated for different growth conditions. The frequency of the P+----P- switch between two steady states was 2.7 x 10(-2). In batch culture the frequency of the P(-)----P+ switch was minimally 2.9 x 10(-2). The results indicate that phase variation and the production of 987P fimbriae by fimbriate cells are under independent physiological control.

Adhesins, Escherichia coli↗
PubMedDate not supplied

Growth phase-dependent variations in the outer membrane protein profile of Brucella melitensis.

Changes in Brucella cell envelope protein profiles were investigated with batch cultures of B. melitensis strain 16M in a 2-litre fermenter. Analysis of expression of outer membrane proteins (OMP) (apparent molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa) and heat-shock protein DnaK (73 kDa) was performed with monoclonal antibodies (mAb) and immunoblotting techniques. Synthesis of the 89-kDa OMP and the heat-shock protein DnaK was invariant during B. melitensis growth. Expression of the 10-, 19- and 36-38-kDa minor OMPs was never detected. Variations in profiles of some OMPs, i.e. 25-27-kDa and 31-34-kDa major proteins and 16.5-kDa minor protein, occurred during growth stages, principally at the end of the exponential growth phase. These variations consisted of shifts in apparent molecular masses for the 25-27-kDa and 31-34-kDa OMPs and of peptidoglycan association for the 16.5-kDa OMP. Therefore, whereas the strong association of major OMPs with peptidoglycan was confirmed, results suggested that the 16.5-kDa minor OMP is also a peptidoglycan-associated protein.

Bacterial Outer Membrane Proteins↗
PubMed2005

High relative content of lysophospholipids of Helicobacter pylori mediates increased risk for ulcer disease.

Helicobacter pylori phospholipase A (OMPLA) degrades bacterial membrane phospholipids to lysophospholipids. High levels of lysophospholipids are associated with higher hemolytic activity, increased release of urease and vacA and better adherence to epithelial cells in vitro. The phospholipase A gene (pldA) displays phase variation due to a slippage in a homopolymeric tract. The aim of this study was to determine if the relative amount of lysophospholipids in the cell wall is associated with ulcer disease, and to further investigate the significance of pldA phase variation. H. pylori isolates of 40 patients were examined. The relative lysophospholipid content of each isolate was determined and the pldA gene was sequenced. The study indicated that H. pylori can regulate its OMPLA activity by phase variation in the pldA gene or by protein level regulation among phase variants in the pldA 'ON' status. We found a significant difference between the relative amount of lysophospholipids of the ulcer group and the non-ulcer group (p=0.022). When the lysophospholipid/phospholipid ratios were compared with outcome, the OR for ulcer disease was 9.0 (95% CI 1.6-49.4; p=0.014). Isolates with a high OMPLA activity are significantly associated with patients with ulcer disease.

Base Sequence↗
PubMed1999

Frequencies of lipopolysaccharide-defined epitopes in Haemophilus influenzae type b and non-typable isolates determined with monoclonal antibodies.

OBJECTIVE: To investigate the frequency of expression and stability of saccharide epitopes in 178 Haemophilus influenzae (39 type b and 138 non-typable) isolates from blood, cerebrospinal fluid, nasopharynx, pharynx, middle ear, conjunctiva, and pleural and bronchial fluid from symptomatic and asymptomatic children using five murine monoclonal antibodies (MAbs, MAHI 3, 4, 6, 8, 10) specific for the oligosaccharide moiety of the lipopolysaccharide (LPS) of H. influenzae, which recognize defined saccharide structures. METHODS: A whole bacteria enzyme immunoassay (EIA) and colony dot immunoblotting were used to determine the frequency of expression and stability of saccharide epitopes in the 178 H. influenzae isolates. RESULTS: Six main groups of strains were differentiated based on the EIA binding pattern with the MAbs: group A, reactive with all five MAbs (MAHI 3, 4, 6, 8 and 10); group B, reactive with four MAbs (MAHI 3, 6, 8 and 10); group C, reactive with three MAbs (MAHI 3, 6 and 8); group D, reactive with three MAbs (MAHI 3, 6 and 10); group E, reactive with two MAbs (MAHI 3 and 10); group F, reactive with MAb MAHI 3. Group B was the most common classification overall. None of the strains remained non-reactive. The frequencies of the binding patterns among the isolates obtained from different sources appeared to be statistically similar in most of the cases. Indications of phase variation of the LPS epitopes were observed with all the MAbs for strains obtained from all clinical sources as evaluated by colony dot immunoblotting. One of the epitopes displayed 22% phase variation, while four other epitopes were variably expressed, with about 50% on-off expression. CONCLUSIONS: This set of MAbs showed 100% reactivity among the isolates, in both EIA and colony dot immunoblotting, and allowed us to differentiate strains based on the LPS phenotype by whole bacteria EIA. Phase variation was indicated among all the isolates, independent of the source of isolation, and for all five MAbs. The LPS of isolates from different clinical sources often expressed some of the epitopes recognized by the MAbs, and most of the LPS phenotypes appeared at similar frequencies among isolates.

Journal Article↗
PubMed1987

Human bactericidal antibody response to meningococcal outer membrane protein vaccines.

Several different meningococcal outer membrane protein vaccines have been prepared and used in human safety and immunogenicity studies. The results of these studies have led to some general conclusions regarding the human antibody response to these vaccines. A review of these conclusions, however, indicates that a number of important questions and problems still need to be addressed. Two of these are the determination of the protective level of bactericidal antibody in human serum and the impact of phase variation of surface antigens on vaccine strategy. Bactericidal assays using intrinsic complement and high concentrations of serum suggest that the level of natural immunity to group B meningococci is quite high, but is increased by vaccination with outer membrane protein vaccine. Phase variation in meningococcal surface antigens including capsule, class 1 protein, class 5 protein, and lipopolysaccharide was demonstrated using colony blotting with monoclonal antibodies. Phase variation resulted in differences in susceptibility to the bactericidal activity of human sera.

Antibodies, Bacterial↗
PubMedDate not supplied

Correction of phase wrapping in magnetic resonance imaging.

In phase reconstruction MR imaging, e.g., for velocity measurement, phase shifts beyond +/- pi radians will "wrap around" to smaller apparent phases. Such large phase shifts could arise either due to large background (non-flow-related) phase variations or due to large velocity-induced phase shifts. For sufficiently smooth phase variation, such discontinuities can be automatically recognized and corrected, thus restoring the correct phase values and extending the effective dynamic range of such phase imaging techniques.

Magnetic Resonance Imaging↗
PubMed1991

The Vlp system of Mycoplasma hyorhinis: combinatorial expression of distinct size variant lipoproteins generating high-frequency surface antigenic variation.

Isogenic populations of Mycoplasma hyorhinis undergo in vitro high-frequency phase variation in the expression of surface lipoproteins; these products also vary markedly in size through changes in periodic protein structure (R. Rosengarten and K.S. Wise, Science 247:315-318, 1990). In this report, we rigorously define three distinct translation products comprising the Vlp (variable lipoprotein) system of M. hyorhinis SK76 and establish parameters of Vlp structural diversity and expression that distinguish the Vlp system from previously described examples of antigenic variation. VlpA, VlpB, and VlpC are prominent amphiphilic membrane lipoproteins characterized by detergent-phase fractionation and metabolic labeling with [35S]cysteine and [3H]palmitate. VlpA is distinguished from VlpB and VlpC by its selective labeling with [35S]methionine; VlpB and VlpC are distinguished by specific epitopes defined by surface-binding monoclonal antibodies (MAbs); a third MAb defines a surface epitope shared by VlpB and VlpC (but absent from VlpA). Each Vlp displays 12 to 30 spontaneous size variant forms comprising a periodic ladder that could also be generated by partial trypsin digestion of individual Vlp size variants. Different periodic intervals within VlpB and VlpC further distinguish these two products structurally. Mycoplasma colony opacity correlates inversely with Vlp size. Each Vlp undergoes independent, oscillating high-frequency phase variation in isogenic populations and can be expressed individually or concomitantly with other Vlps in a noncoordinate manner. All seven possible combinations of these three products were observed; however, no variants were found that lacked a Vlp. High-frequency size variation of each Vlp superimposed on combinatorial diversity in Vlp expression yields greater than 10(4) possible structurally distinct Vlp mosaics, of which 104 were documented along with 24 of 42 possible transitions among the seven Vlp combinations. In addition to these features, VlpA, VlpB, and VlpC were specifically recognized by serum antibodies from swine with experimental M. hyorhinis SK76-induced arthritis, indicating expression and immunogenicity of Vlps in the natural host. The structure and variation of Vlps and their known involvement in MAb-mediated modulation of mycoplasma-infected host cell properties and mycoplasma killing are discussed in relation to the surface architecture and adaptive potential of the wall-less mycoplasmas.

Animals↗