Mycoplasma Pneumoniae Infection Interferes With Sphingolipid Metabolism In Human Lung Carcinoma A549 Cells

Mycoplasma pneumonia(M.pneumoniae)is a human pathogen which is able to cause community acquired respiratory infections in adults and children.Besides respiratory illness,as many as 25%persons infected with M.pneumoniae experienced extrapulmonary complications such as encephalitis,polyradiculitis,which is sufficiently severe to require hospitalization.Moreover,M.pneumoniae has recently been linked to asthma.Although considerable progresses have been made over the years for the study of M. pneumoniae pathogenesis on cytadherence,cytotoxicity and inflammation,the molecule mechanisms underlying M.pneumoniae pathogenesis still remain elusive.With the advent of "lipidomics",in which individual lipid components can be analyzed by mass spectrometry(MS),various unexpected findings on lipid mediators of inflammation led to elucidate bacterial pathogenesis at a lipid level.More recently, studies have begun to implicate a class of lipids,the sphingolipids,are important signaling molecules involved in many cellular functions including cell proliferation, differentiation,apoptosis,stress response,inflammation.Besides,the role of sphingolipids in infectious diseases has also been recognized,For instance,host defense against pathogens also requires ceramide-enriched membrane platforms.On the other hand,many pathogens use host membrane sphingolipids as receptors,and lipid rafts can be used as platforms for infection signaling and entry of intracellular parasites.M.pneumoniae is one of the smallest organisms capable of autonomous growth.As lacking the genes needed for synthesizing certain lipids,such as cholesterol,it has the ability to incorporate them from the surrounding environment.M.pneumoniae contain a few sphingolipids,but it could use exogenous sphingolipids(from the surrounding environment,such as host)to synthesize glycosphingolipids.Molecular mimicry has been suggested to play a vital role in M.pneumoniae infection-induced acute motor axonal neuropathy Guillain-Barré(GBS)or Fisher(FS)syndrome,in which antibodies targeted to gangliosides(one of the glycosphingolipids species)are responsible for these autoimmune diseases.However,to date there has been no systematic analysis of the sphingolipid profile of M.pneumoniae,even less for the effects of M.pneumoniae infection on host sphingolipid metabolism.In this study,we first adopted a lipidomic approach to study the effects of M.pneumoniae infection on the sphingolipid profile of human lung carcinoma A549 cells,and then further studied the effects of M.pneumoniae infection on sphingolipid metabolism in A549 cells.ResultsComparison of sphingolipids profile in M.pneumoniae with uninfected A549 cells by MALDI-TOF MSWe first apply MALDI-TOF MS to identify the sphingolipids profile in M. pneumoniae,and compare with that of A549 cell.It is found that M.pneumoniae and A549 cells had many sphingolipids molecules in common,yet peaks 507.8,511.1, 549.7,574.5,729.6,751.5,and 833.3 were only present in M.pneumoniae.Therefore, we defined these 7 peaks as markers of M.pneumoniae sphingolipids.On the other hand,peaks 508.9 and 518.8 were only found in A549 cells.Infection of M.pneumoniae induced significant change of cellular sphingolipid compositionA549 cells were infected by M.pneumoniae at the concentration of 1 CFU/cell, 10 CFU/cell and 100 CFU/cell.Sphingolipids were extracted from the infected and uninfected cells after 2 h,12 h,and 24 h infection,and analyzed by MALDI-TOF-MS. it was found that M.pneumoniae infection dramatically changed ceramide and sphingomyelin composition in cells.For example,2 h after 1 CFU/cell infection,the relative amounts of some ceramide and sphingomyelin species were changed.At 12 and 24 h,however,there was the generation of new ceramide species.The effects of 10 CFU/cell were stronger than those of 1 CFU/cell,as at 2 h post-infection there was already the induction of new ceramide species.Interestingly,the effects of 100 CFU/cell were most dramatic only at 24 h,with the generation of many new ceramide and sphingomyelin species.Thus,it appears that the effects of M.pneumoniae infection on cellular sphingolipid metabolism depend on the different infection doses as well as the infection times.M.pneumoniae infection affects serine palmitoyltransferase expression in A549 cellsSince dramatic change happened in cellular sphingolipid composition due to infection, we examined the mRNA levels of two key enzymes in the metabolism of sphingolipids-acid sphingomyelinase(ASM)and serine palmitoyltransferase(SPT)-in the infected and uninfected cells by RT-PCR.SPT is the rate-limiting enzyme in the de novo synthesis pathway for ceramide,while ASM is responsible for hydrolyzing sphingomyelins to ceramides.The statistical analysis showed that the mRNA level of SPT decreased significantly after 24h infection while under other circumstance the expression of ASM and SPT remained unchanged.Clustering of ASM on cell plasma could be induced by infection of M. pneumoniae at certain concentration and time pointWe found that 2h and 24h infection did not induce ASM clustering(data not shown).After 12h infection,ASM clustering on lipid rafts can be induced by 1CFU/cell and 10CFU/cell M.pneumoniae and the higher infection concentration induced ASM clustering in more cells,but no clustering was observed under infection at 100CFU/cell.Conclusion:1.We first identify the sphingolipids profile of M.pneumoniae by applying lipidomic approach,which will improve our understanding on the M. pneumoniae glycolipids connected with autoimmune diseases.2.Infection of M.pneumoniae with host cell can lead to the production of new species of ceramides as well as the ASM clustering on lipid rafts,this event may contribute to the release of several proinflammatory mediators by the host.