Specific binding of Burkholderia pseudomallei cells and their cell-surface acid phosphatase to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2).

Specific binding between bacterial cells and host tissue is an early step of the pathogenesis of infection. Burkholderia pseudomallei cells, the causative micro-organisms of melioidosis, were demonstrated to bind specifically to tissue glycolipids (asialo GM1 and asialo GM2) by solid phase binding assay on thin layer chromatograms. The detection limit was around 400 pmol of the glycolipids. Acid phosphatase purified from the culture filtrate of B. pseudomallei was tested for such binding properties, and the same results were obtained. According to our previous studies, the enzyme is a glycoprotein located on the cell surface, and hydrolysed tyrosine phosphate most actively among the substrates so far tested. The mode of binding between the enzyme and the glycolipids was analyzed by comparison of binding levels among three samples different in protein content, sugar content and specific phosphatase activities per protein and sugar residue. The results suggested the possibility of a receptor-ligand relationship between the bacterial enzyme and the host-cell glycolipids (asialo GM).

Evolution of cell-surface acid phosphatase of Burkholderia pseudomallei.

Acid phosphatase active fractions were obtained from cell-free extract, outermembrane fraction and culture filtrate of Burkholderia pseudomallei by column chromatography with sepharose 6B and DEAE cellulose. The comparison of the elution patterns of protein, sugar and enzymatic activity among these three components suggested that the enzyme is a glycoprotein evolving from premature proteins through glycosylation and that the enzyme is translocated during glycosylation from the cytoplasm to the outer membrane and finally excreted into the environment. When tunicamycin, a glycosylation inhibitor, was added to the culture, the peaks of sugar and enzymatic activity were lowered concomitantly leaving the protein peak unchanged in the elution pattern of the culture filtrate. The affinity of the bacterial surface to antienzyme sera was demonstrated by immuno-fluorescence microscopy.

Affinity and response of Burkholderia pseudomallei and Burkholderia cepacia to insulin.

The cells of Burkholderia pseudomallei, B. cepacia and Pseudomonas aeruginosa grown on agar plates were stained with fluorescently-labeled insulin. The former two species were stained positively indicating insulin binding but P. aeruginosa was not. Insulin exposure reduced phospholipase C and acid phosphatase activities of B. pseudomallei but did not affect those enzymatic activities of B. cepacia in the employed experimental conditions. It is suggested that B. pseudomallei have insulin receptors which may be associated with a signal transfer system involving phospholipase and protein tyrosine phosphatase.

Multifactorial pathogenic mechanisms of Burkholderia pseudomallei as suggested from comparison with Burkholderia cepacia.

With the purpose to elucidate the pathogenesis of disease due to Burkholderia pseudomallei some biological and biochemical properties of this species were studied in comparison with B. cepacia, since the difference in the level of virulence between the two species is remarkable despite of their toxonomic closeness. B. pseudomallei was distinct from B. cepacia in the capability to grow under anaerobic conditions, with positive nitrate respiration, excretion of high-molecular polysaccharides into liquid culture, and cytotoxicity against cultured tissue cells. From these observations together with our previous finding that B. pseudomallei can grow and survive in an acidic environment, we suggest multifactorial mechanisms for the pathogenesis of melioidosis due to B. pseudomallei.

Separation of antigenic glycoprotein fractions from cell-free homogenate of Pseudomonas pseudomallei and characterization as tyrosine phosphatase.

Cell-free extracts were prepared from Pseudomonas pseudomallei cells by freezing-thawing, sonication, and differential ultracentrifugation. The extracts were subjected to column chromatography with DEAE-sepharose to obtain glycoprotein fractions. The fractions showed acid phosphatase activity to p-nitrophenyl phosphate, tyrosine phosphate, serine phosphate, but not to threonine phosphate. They were highly antigenic when tested by immunofluorescence assay with the sera of melioidosis patients.

Effects of tunicamycin on the pH-activity pattern of acid phosphatase in Pseudomonas pseudomallei.

The liquid culture of Pseudomonas pseudomallei shows a complex feature in in the pH-activity pattern of acid phosphatase, not a single peak curve. There was an evident tendency that the higher activity shifted to the higher pH range with the growth of culture. The culture in the presence of tunicamycin (20 micrograms/ml) showed a decreased activity selectively in the higher pH range, while the activity in the lower pH was more heat-labile. The bacterial cells grown on agar plates containing tunicamycin were more heat-labile than the untreated control cells. The glucosidase-treatment reduced the enzymatic activity (of the phosphatase-active fractions from the living cells) with the shift of the optimum pH to lower pH. These observations together with some collateral findings suggest that the pH-activity pattern of acid phosphatase in P. pseudomallei is associated with the development of precursor enzyme proteins to mature glycoproteins.

Application of indirect immunofluorescence microscopy to colony identification of Pseudomonas pseudomallei.

Indirect immunofluorescence microscopy was used as a colony identification method of Pseudomonas pseudomallei isolates. The antisera against lipopolysaccharide and protein fractions of P. pseudomallei were prepared in guinea pigs and rabbits. With these antisera and fluorescence-labelled anti-guinea pig IgG and anti-rabbit IgG prepared in sheep (goat), indirect immunofluorescence microscopy was conducted on the colonies of P. pseudomallei and other species of bacteria. The overall results indicated that this method is efficient, rapid and specific for identification of P. pseudomallei colonies from clinical specimens.

Use of endotoxin antigens in enzyme-linked immunosorbent assay for the diagnosis of P. pseudomallei infections (melioidosis).

An enzyme-linked immunosorbent assay (ELISA) with endotoxin preparations of P. pseudomallei as antigen was developed for detection of IgG antibodies specific to melioidosis. Forty-seven sera of bacteriologically confirmed melioidosis patients, 55 non-melioidosis sera and 50 sera of healthy blood donors from non-endemic areas were subjected to this assay in comparison with indirect hemagglutination assay (IHA). The data were treated by receiver operating characteristics analysis. The sensitivity, specificity and accuracy in this ELISA were 95.7%, 94.2%, and 94.7%, respectively, with cut-off value of OD = 0.312 at 490 nm. Meanwhile, those in IHA were 81.0%, 91.4%, and 88.1%, respectively, with a cut-off value of > or = 1:160. From these results, the ELISA was judged to be more reliable than IHA as the seroassay for diagnosis of melioidosis.

Species-differences in the pH-activity pattern of acid phosphatase in the family Enterobacteriaceae.

Nonspecific phosphatase activities were surveyed comparing major species of the family Enterobacteriaceae. The strains were subjected to a whole cell assay system with paranitrophenyl phosphate as substrate over a wide pH range and with a standardized number of bacterial cells. The overall results suggest that the general shape of the pH activity curve and the location of peaks (pH optimum) can be employed as a supplementary criterion to characterize species of Enterobacteriaceae.

Immunofluorescence, enzyme-linked immunosorbent assay, particle agglutination and western blot for the detection of antibody to human immunodeficiency virus type 1.

Immunofluorescence assay (IFA) has been applied for detection of antibody to human immunodeficiency virus type 1 (HIV-1). To compare the IFA with an enzyme-linked immunosorbent assay (ELISA) and particle agglutination (PA), we examined the antibody response to HIV-1 in 475 sera from AIDS, PGL and ARC patients as well as several risk groups and healthy persons by three methods. The positive results by any methods were confirmed by western blot (WB). The results by all methods were well correlated on the sera from 45 asymptomatic male homosexuals and 70 female prostitutes. There were some false positive results by ELISA in the sera from prisoners and healthy persons. Four sera from drug abusers were positive only by PA and IFA and were negative by ELISA. All were WB-inconclusive. Particle agglutination and IFA results were compared with western blot analysis on 208 ELISA-positive sera. All IFA-strongly positive sera (84%) were positive by western blot. The sera with weakly positive, negative and inconclusive results by IFA (16%) were possibly any of positive, inconclusive or negative by western blot. By PA, 200 of 208 (97%) sera were PA-positive and 1% of these sera were WB-inconclusive while the PA-negative sera were either negative or inconclusive by western blot. These results suggested that PA is a simple and sensitive method for screening of HIV-1 antibody while IFA could be a primary confirmatory test and western blot would then be used for confirming any IFA-negative or inconclusive results.