The Fat and the Ugly: A Proteomics Approach to Dissect Lipid Droplet-Chlamydia Interactions

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

Durham, Carolina del Norte

Congreso; North Carolina American Society for Microbiology 2009 Meeting; 2009

American Society for Microbiology (North Carolina Branch)

2009 NC ASM Meeting – Short talk abstractThe obligate intracellular pathogen Chlamydia trachomatis (CT) is the most common cause of sexually transmitted infections and the leading cause of preventable blindness worldwide. CT infects epithelial cells and multiplies within a membrane-bound vacuole or “inclusion”. Lipid droplets (LD), the main store of neutral lipids in eukaryotic cells, constitute dynamic, ER-derived organelles consisting of a core of cholesterol esters or triacylglycerols, surrounded by a monolayer of phospholipids and LD-associated proteins. Previous evidence from our laboratory shows that CT targets LDs, inducing an expansion of these organelles, their recruitment around the inclusion and ultimately their wholesale transport into the lumen of the inclusion. These findings represent the first example of mammalian LD functions being co-opted by a bacterial pathogen and highlights novel aspects of chlamydial cellular microbiology.  To define the molecular basis for Chlamydia-LD interactions, LDs from infected and uninfected cells were purified, LD-protein extracted and 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 identification and absolute quantitation of peptides detected in the samples. 311 proteins were identified in LDs, including most of the already recognized LD-associated proteins (i.e., the PAT family, lipid metabolism-related proteins, Rab GTPases, chaperones and cytoskeleton proteins). A highly restrictive algorithm allowed the identification and reliable quantification of a subset of 162 proteins in all the processed samples (2 biological replicates, 6 runs each, for LD-uninfected, LD-20 hpi and LD-40 hpi). These proteins ( >90% of the samples total mass), were categorized based on protein function/localization and changes in LD-protein levels from infected vs. uninfected cells were analyzed. In LDs from Chlamydia-infected cells, lipid-metabolism proteins were increased whereas a large number of ER chaperones, MHC class I and PDI were decreased. Considering that lipid acquisition is known to be essential for bacterial replication and that PDI and MHC class I are essential for optimal antigen processing/presentation, we propose that CT targets LD to co-opt lipid transport and ER functions. Additionally, the association of MHC-class I molecules to LDs supports a possible intersection between these organelles and the immune response. This is to our knowledge, the first quantitative determination of these organelles’ proteome and the first comparative analysis of the LD proteome in the context of a bacterial infection.