Molecular cloning and mutagenesis of a DNA locus involved in lipooligosaccharide biosynthesis in Haemophilus somnus

Haemophilus somnus undergoes antigenic and structural phase variation in its lipooligosaccharide (LOS). A gene (lob-1) containing repetitive 5'-CAAT-3' sequences that may, in part, contribute to phase variation was cloned and sequenced (T. J. Inzana et al., Infect. Immun. 65:4675-4681, 1997). We have now identified another putative gene (lob-2A) immediately upstream from lob-1. Lob-2A contained homology to several LOS biosynthesis proteins of the family Pasteurellaceae and the LgtB and LgtE galactosyltransferases of Neisseria meningitidis and N. gonorrhoeae. Unlike lob-1, lob-2A contained 18 to 20 5'-GA-3' repeats 141 bp upstream of the termination codon as determined by PCR amplification of DNA from individual colonies. Twenty repeats were most common, but when 19 5'-GA-3' repeats were present a stop codon would occur 1 bp after the last 5'-GA-3' repeat. A 630-bp SalI-BsgI fragment within lob-2A was deleted, and a kanamycin resistance (Km(r)) gene was inserted into this site to create pCAATDeltalob2A. Following electroporation of pCAATDeltalob2A into H. somnus 738, several allelic exchange mutants were isolated. The LOS electrophoretic profile of one mutant, strain 738-lob2A1::Km, was altered, and the phase variation rate was reduced but phase variation was not eliminated. A variant with 19 5'-GA-3' repeats in lob-2A had an LOS profile similar to that of 738-lob2A1::Km, suggesting that lob-2A was turned off in this phase. Nanoelectrospray mass spectrometry (nES-MS) and nuclear magnetic resonance spectroscopy showed that 738-lob2A1::Km was deficient in the terminal betaGal(1-3)betaGlcNAc residue present in parent strain 738. Mutant 738-lob2A1::Km was significantly more sensitive to the bactericidal action of normal bovine serum and was less virulent in mice than was parent strain 738. When H. somnus 129Pt was electrotransformed with shuttle vector pLS88 containing lob-2A, its LOS electrophoretic profile was modified and additional N-acetylhexosamine residues were present, as determined by nES-MS analysis. These results indicated that lob-2A may be an N-acetylglucosamine transferase involved in LOS biosynthesis and phase variation and that LOS structure is important to H. somnus virulence.     

Malaria transmission and naturally acquired immunity to PfEMP-1

Why there are so few gametocytes (the transmission stage of malaria) in the blood of humans infected with Plasmodium spp. is intriguing. This may be due either to reproductive restraint by the parasite or to unidentified gametocyte-specific immune-mediated clearance mechanisms. We propose another mechanism, a cross-stage immunity to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1). This molecule is expressed on the surface of the erythrocyte infected with either trophozoite or early gametocyte parasites. Immunoglobulin G antibodies to PfEMP-1, expressed on both life cycle stages, were measured in residents from an area where malaria is endemic, Papua New Guinea. Anti-PfEMP-1 prevalence increased with age, mirroring the decline in both the prevalence and the density of asexual and transmission stages in erythrocytes. These data led us to propose that immunity to PfEMP-1 may influence malaria transmission by regulation of the production of gametocytes. This regulation may be achieved in two ways: (i) by controlling asexual proliferation and density and (ii) by affecting gametocyte maturation.     

Vascular lesions in testes associated with male infertility in Cameroon

Summary Testicular biopsies in 40 of 41 infertile males with severe oligospermia in Cameroon presented massive subendothelial fibrinoid deposits in the small and medium sized vessels. Fibrinogen, complement and IgM were demonstrated in these deposits by immunofluorescence. Evidence strongly suggestive of parasitic testicular involvement was also observed in 2 cases.It is postulated that the fibrinoid deposits are the result of repeated formation and deposition of circulating immune complexes by reaction of antibodies with antigens. These antigens could be of various origins and in the cases described here they could be derived from living or dying parasites in the region. The accumulation and incorporation of the fibrinoid deposits may lead to vascular stenosis resulting in chronic ischaemia, tubular atrophy and fibrosis, and finally oligospermia.     

Improved chamber for the isolation of anaerobic microorganisms.

A small portable chamber for the recovery of anaerobic bacteria is described. This rigid chamber is constructed of clear acrylic with dimensions of 30 inches (ca. 76.2 cm) wide, 18 inches (ca. 44.7 cm) deep, and 18 inches (ca. 44.7 cm) high. Conventional bacteriological techniques can be used inside the chamber to efficiently isolate strict anaerobic organisms. An adapter allows the attachment of a standard anaerobic jar to the outside of the chamber. The jar can be used to store reduced media. Once the jar is attached to the chamber and the media is removed to the interior of the chamber, the jar is available to receive inoculated media. The anaerobic jar can then be removed from the chamber, without contaminating the jar or chamber with oxygen, and be placed in a conventional 37degreesC incubator. This chamber also allows the microbiologist to process cultures without wearing gloves as was necessary with previous anaerobic chambers. Air-tight latex rubber sleeves seal around the microbiologists arms and to the armport flange of the chamber to prevent the introduction of oxygen into the chamber. Anaerobic conditions are maintained by circulating a 80% N2, 10% H2, 10% CO2 gas mixture through alumina pellets coated with palladium. This study indicates that anaerobic conditions obtained in this chamber are sufficient for recovery of obligate anaerobes.     

Homozygosity for the predominant Cys282Tyr mutation and absence of disease expression in hereditary haemochromatosis.

A candidate gene for hereditary haemochromatosis, HLA-H, has recently been presented. Two missense mutations in the HLA-H gene sequence are predicted to account for nearly 90% of all cases of the disease. The aim of this study was to correlate the presence of these missense mutations with the expressivity of the disease, as assessed by standard biochemical evaluation of serum iron parameters. Detection of the known mutations in haemochromatosis, Cys282Tyr and His63Asp, was undertaken in a large pedigree showing variable expression of the disease in successive generations. In three sibs with overt disease (one male, two female, aged 50 to 53 years), homozygosity for the predominant G to A transition (Cys282Tyr) in HLA-H was detected. However, homozygosity for this mutation was also detected in an asymptomatic male sib, aged 50, harbouring an identical genotype. The finding of an asymptomatic homozygous Cys282Tyr subject, haplo-identical to affected sibs, indicates that clinical expression of symptomatic disease is variable, even in middle aged Cys282Tyr homozygotes. This has profound implications for the future use of genetic screening for haemochromatosis.     

The transparency of the mammalian cornea

1. A theoretical and experimental analysis of the relationship of the corneal stromal ultrastructure with light transmission has been made in an attempt to resolve recent contradictory explanations of corneal transparency.2. The spatial distribution of collagen fibrils in electronmicrographs of rabbit corneal stroma has been analysed in terms of a radial distribution function. The results indicate the presence of local order extending to at least 200 nm from individual fibrils.3. The observed spatial distribution of the collagen fibrils was used as a basis to compare the theoretically derived and the experimentally determined values of light transmission. It has been found that the transparency of the normal cornea may be explained by the quasi-random structure revealed by the electronmicroscope.4. Histograms of the collagen fibril diameter in normal rabbit corneal stroma revealed the range to be 12.5-32.5 nm and the mean value to be approximately 20 +/- 1.5 nm. Corneal swelling did not change the collagen fibril diameter significantly.5. It is concluded that the size and distribution of collagen fibrils revealed in electronmicrographs are consistent with the observed transparency of normal stromas.6. A marked heterogeneity in the spatial distribution of collagen fibrils was found in the swollen cornea. This is qualitatively consistent with the observed decrease in transparency.     

Isolation of Coccidioides immitis F antigen by immunoaffinity chromatography with monospecific antiserum.

Detection of antibody to Coccidioides immitis F antigen is of proved value in the diagnosis of coccidioidomycosis. This antibody is demonstrable by use of an immunodiffusion assay with reference coccidioidin antigen and antiserum to C. immitis. Using a combination of lectin affinity and immunoaffinity chromatography, we isolated the F antigen from coccidioidin and prepared monospecific antibody to the purified antigen. The availability of these reagents will enable the development of a sensitive and specific assay for detecting serologic reactivity to this antigen.     

Analysis of the MTHFR 1298A-->C and 677C-->T polymorphisms as risk factors for neural tube defects

The thermolabile variant (677TT) of methylenetetrahydrofolate reductase (MTHFR) is a known risk factor for neural tube defects (NTDs). The relationship between a second MTHFR polymorphism (1298A→C) and NTD risk has been inconsistent between studies. We genotyped 276 complete NTD triads (mother, father and child affected with an NTD) and 256 controls for MTHFR 1298A→C. Our findings do not support a role for the 1298A→C polymorphism in NTDs (OR 0.85 (95% CI 0.49–1.47), p= 0.55), nor do we observe a combined effect with the 677C→T polymorphism. Electronic Publication