LI1035 Of Lawsonia Intracellularis Targets MAPK Pathway And Regulates Actin Organization In Yeast And Mammalian Cells

Lawsonia intracellularis is an obligate intracellular Gram-negative bacterium that has been identified as the etiological agent of the contagious disease proliferative enteropathy(PE)in a wide range of animals,mainly pigs.The genome sequence of L.intracellularis indicates that this bacterium possess a type III secretion system(T3SS),which may assist the bacterium during cell invasion and host innate immune system evasion and could be a mechanism for inducing cellular proliferation.However,the effectors secreted by the T3SS(T3Es)of L.intracellularis have not been reported.T3 Es often target conserved eukaryotic cellular processes,and yeast is an established and robust model system in which to reveal their function.Via growth inhibition screening of an ordered array of Saccharomyces cerevisiae strains expressing the hypothetical genes of L.intracellularis,LI1035 was identified as the first putative effector that inhibits yeast growth.The LI1035-induced growth inhibition was rescued in two of the 14 mitogen-activated protein kinase(MAPK)yeast haploid deletion strains,suggesting that LI1035 interacts with the components of the MAPK pathway in yeast.Phosphorylation assays confirmed that LI1035 inhibits MAPK signaling cascades in yeast and mammalian cells.Actin staining assays revealed that LI1035 regulates actin organization in yeast and mammalian cells.Taken together,these results indicate that LI1035 alters MAPK pathway activity and regulates actin organization in the host.These findings may contribute to the understanding the pathogenesis of L.intracellularis and support the use of yeast as a heterologous system for the functional analysis of pathogen-specific gene products in the laboratory.To facilitate to determine the localization of a protein in yeast,the yeast reporter strains that localize to specific structures were developed for co-localization studies.The yeast reporter strains were constructed by chromosomal integration of brighter and most yeast-optimized version of the red fluorescent protein,called RedStar at the carboxy terminal end from their endogenous promoters and verified by colony PCR and analyzed by fluorescence microscopy.The co-localization experiments of DNA-binding dyes(DAPI),Mitotracker(mitochondria)and SipA in Salmonella enteritidis with the corresponding reporter strain were also performed.These yeast reporter strains containning indicators for the vacuole,endoplasmic reticulum,mitochondria,peroxisomes,lipids,endosomes,and the Golgi apparatus were constructed successfully.Our studies provide a basic tool for observing the dynamic changes of organelles and identifying the localization of unknown proteins in yeast.