Changes in the Lipid Droplets Proteome in Response to Bacterial Infection

HECTOR A. SAKA; J WILL THOMPSON; LAURA G. DUBOIS; M. ARTHUR MOSELEY; RAPHAEL VALDIVIA

Steamboat Springs, Colorado, USA

Conferencia; FASEB Summer Research Conferences 2010; 2010

Federation of American Societys For Experimental Biology (FASEB)

Lipid droplets (LD) are dynamic, ER-derived organelles that constitute the main store of neutral lipids in eukaryotic cells. They are primarily composed of cholesterol esters and triacylglycerols, surrounded by a monolayer of phospholipids and LD-associated proteins. The obligate intracellular pathogen Chlamydia trachomatis (CT), the most common cause of sexually transmitted infections and the leading cause of preventable blindness worldwide, recruits and translocates LDs into the lumen of the chlamydial parasitopherous vacuole.  To define the molecular basis for this manipulation of LD by Chlamydia, LDs associated proteins from infected and uninfected HeLa cells were analyzed by mass spectrometry. A state of the art technique for proteomics analysis by bottom-up LC-MS/MS and LC-MSE (Waters nanoAcquity/Synapt-HDMS instrumentation) was used, allowing for the identification and absolute quantitation of 242 proteins (2 or more peptides to match, detected in at least 3 independent runs), including most of the already recognized LD-associated proteins (i.e., perilipins, lipid metabolism-related proteins, Rab GTPases, chaperones and cytoskeleton proteins). The expression levels of a subset of 205 proteins (2 biological replicates, 6 runs total, relative standard deviation less than 50%, for LD-uninfected, LD-20 hpi and LD-40 hpi) were calculated, allowing an assessment of changes in the LD proteome in response to infection. PLIN3 (Tip47) was the most abundant LD protein, representing by itself ~ 11% of the total mass.  Perilipins, lipid metabolism proteins, chaperones and cytoskeleton proteins were dominant, representing ~ 61% of the total mass. In response to infection, lipid-metabolism proteins were found to be increased in LDs whereas a large number of ER chaperones, MHC class I and PDI were decreased. We propose that CT targets LD to co-opt lipid transport.  In addition, the association of immune-related molecules with LDs indicated a possible intersection between these organelles and innate immune responses. Because interferon (IFN) responses are important in chlamydial containment, we tested how the LD proteome changed upon IFN-gamma treatment in MEF (mouse embryo fibroblast) cells, where Chlamydia induces a strong IFN response. Surprisingly, a number of p47 GTPases and other IFN-induced proteins were highly enriched in LDs upon IFN-gamma treatment. Because these proteins are important to contain Chlamydia and other intracellular pathogens it is possible that LDs may participate in the cell autonomous immune response against intracellular pathogens.  As such, the translocation of LDs into the Chlamydia parasitopherous vacuole may prevent the proper function of effector proteins of the interferon response and represent a mechanism of immune evasion by this pathogen.