[Phospholipase C activity and alteration of intracellular free Ca2+ levels during internalization of Leptospira interrogans].
To determine the effects of leptospiral strains with different virulence on intracellular free Ca(2+)level and its relation with phospholipase C (PLC) activity of L.interrogans.



L.interrogans-j infection cell modals were established with Vero and J774A.1 cell lines. Vero and J774A.1 cells were co-incubated with L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 (strong virulence) and serogroup Pomona serovar pomona strain 56608 (weak virulence) and L.biflexa serogroup Samaranga serovar patoc strain Patoc I (non-virulence). Intracellular free Ca(2+)levels were detected by laser scanning confocal microscopy with specific fluorescence labeling of fluoj3/AM. Using [(3)H] PIP2 as the substrate, the PLC activities in the culture supernatant, and cytoplasma and cytomembrane of the three strains of Leptospira were measured by isotope assay.



The baseline intracellular free Ca(2+)levels in the normal Vero and J774A.1 cells were (102.3+/-8.2)% and (105.9+/-7.3)%,respectively. The fluorescence intensity in the two cell lines incubated with L.biflexa strain Patoc I were fluctuated in range of (102.3+/-8.2)%approximate, equals(102.2+/-8.3)% during the observation period. The intracellular free Ca(2+)levels in the two cell lines infected with L.interrogans strain 56601 showed elevation with double peak patterns, with first peaks of (430.5+/-35.7)%, (747.5+/-18.5)% and the second peak of (380.6+/-17.4)%, (804.6+/-22.4)%, respectively. When the cells were infected with L.interrogans strain 56608, the intracellular free Ca(2+)levels were rising slowly with a single slope-like pattern, with the maximal of (235.0+/-19.3)% in Vero cells and (402.4+/-17.4)% in J774A.1 cells, which were significantly lower than those in the cells infected with L.interrogans strain 56601 (P<0.01). The culture supernatants, and cytoplasma and cytomembrane proteins of all three strains displayed PLC activities (P<0.05).



The cells infected with L.interrogans of different virulence show distinct intracellular free Ca(2+)levels and peak patterns. The different host cell lines can affect the intracellular free Ca(2+)levels, which is not related to the PLC activity in the leptospiral strains.
Wang
Huan-ping
HP
Department of Medical Microbiology and Parasitology, College of Medicine, Zhejiang University, Hangzhou 310031, China.
Yan
Jie
J
Li
Li-wei
LW
Mao
Ya-fei
YF
Li
Shu-ping
SP
Luo
Yi-hui
YH
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004705:Endocytosis; D006801:Humans; D007921:Leptospira interrogans / Q000201:enzymology* / Q000472:pathogenicity; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology*; D010738:Type C Phospholipases / Q000378:metabolism*; D014709:Vero Cells; D014774:Virulence
D010738:Type C Phospholipases; D002118:Calcium