Legionella pneumophila eukaryotic-like effector LegK3 inhibits growth of Saccharomyces cerevisiae and modulates its vesicle trafficking pathway

Abstract:

Abstract:［Objective］ To study biochemical functions of the Legionella pneumophila eukaryotic-like effector protein LegK3，the budding yeast Saccharomyces cerevisiae was used as an alternative host in which growth defect induced by the ectopic expression of LegK3 was assessed． ［Methods］ Using genomic DNA of the L. pneumophila strain Lp02 as template，we respectively amplified and inserted the ORF sequences of legK3，ralF or lidA into the plasmid pESC-HK to yield the ectopic-expression plasmids． Then，the recombination plasmids were transformed into the yeast strain W301-1A．With 2% -galactose induction，growth defect and carboxypeptidase Y (CPY) delay were determined simultaneously. In parallel，total yeast proteins before or after induction were extracted and subjected to Immunoblot assay. For detecting the expression of effector proteins or determining CPY delay，anti-c-myc or anti-PGK/anti-CPY antibodies were utilized respectively．［Results］The expression of LegK3 resulted in visible growth defect in yeast cells，together with obvious retard in CPY processing．［Conclusion］L．pneumophila eukaryotic-like effector LegK3 might target and interfere with the vesicle-trafficking pathways，thereby to inhibit the growth and division of host cells．