Shigella flexneri strains harboring large-sized plasmid(140 megadalton, Mdal)were subjected for the study of the virulence-related properties.
Four strains of HeLa-invasive Shigella were found to have this large plasmid. This plasmid was confirmed to be strongly related to the invasiveness into HeLa cell since that mutants which lost 140 Mdal plasmid and had 57 to 95 Mdal sized plasmids
instead were unable to invade HeLa cell.
Reliability and efficiency of dye-binding by Shigella strains as a selective marker for the detection of virulent strains were evaluated. When tested on tested on agar plates supplemented with Congo red, basic fuchsin, and crystal violet,
respectively,
all virulent strains (HeLa invasive) showed prominent binding with dyes whereas a part of non-virulent strains occasionally bound with dyes, moreover, the degree of binding seemed to be dependent on the concentration of dyes supplemented. Ability
of
dye-binding was also studied in liquid solutions supplemented with same dyes. Virulent strains showed greater binding with dyes than non-virulent strains but these results were statistically insignificant. The longer the strains reacted with
dyes,
the
more consumption of dyes were observed. On the contrary, binding of crystal violet was rather lower after 24 hours than after 1 hour of reaction.
Beware of the fact that invasiveness into HeLa cell of Shigella could be associated with the outer membrane proteins of Shigella, membrane protein fractions were studied by SDS-PAGE after extraction. As preliminary test, various staining
conditions
were
applied to detect more detailed protein fractions in gel. Obviously there great difference between staining methods and silver staining of gels was more excellent (approxim. 10X) than coomasie brilliant R-250 staining.
Total protein fractions of the S. flexneri 85DH38 showed twelve distinctive bands when gel was stained with silver after SDS-PAGE but only five bands were visible after staining with coomasie brilliant R-250. The molecular weight of twelve bands
each
was 180 kilodalton(kd), 120kd, 100ke, 78kd, 64kd, 55kd, 47kd, 45kd, 43kd, 39kd and 38kd in decreasing order. Among sodium-lauryl-sarcosyl(SLS)-soluble fractions, most distinctive band was the fraction of 100kd, Besides this band, there were five
bands
(180kd, 78kd, 64kd, 55kd and 45kd). In SLS-insoluble fractions, there were three clearly visible bands(47kd, 39kd and 38kd) and these were thought to be porins. And also additional two bands (43kd and 12.8kd) could be observed.
As contrast with 85DH38 strains, S. flexneri 78-b1 showed only seven bands in total protein fractions. Moreover only one band(100kd) was observed in SLS-soluble fractions and three(47kd, 39kd and 12.8kd) were visible in SLS-insoluble fractions.
Electric current during gel electrophoresis possibly affected the result. Additional five visible bands in the gel which was performed under 20mA were invisible in the gel done under 30mA. Molecular sizes of above five bands ranged 10 to 20kd.
Temperature during bacterial cell culture did not influence to the membrane protein fractions regardless of the presence of Congo red.
The very concept of the virulence of Shigella was plasmid-associated HeLa invasiveness, implying that there could be a significant difference in membrane protein fractions in Shigella strains.
|