Deciphering the molecular mechanisms of sterol lipid trafficking in bacteria

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Project summary/abstract Sterols lipids, including cholesterol, are important for mammalian cell physiology. These molecules modulate the fluidity of biological membranes and are therefore implicated maintaining membrane integrity, stress tolerance, fusion events, etc. Sterols are also involved in intra- and intercellular signaling and are trafficked to sub-cellular membranes. Whereas decades of research have provided molecular insights into eukaryotic sterol synthesis, transport, regulation, and function, similar understanding of sterols is lacking for bacteria and archaea. While it is thought that archaea do not make or use sterols, some bacteria do make and transport sterols; many others are known to engage with sterols produced by eukaryotes. These bacteria include the pathogenic spirochetes (Borrelia burgdorferi, Treponema pallidum), Mycobacteria, Chlamydia, Rickettsia, and gut microbiota. For pathogens, the acquisition of sterols from the host is critical as they colonize and construct their cell envelopes. For gut microbes, interactions with cholesterol can alter the host lipid metabolism, thereby contributing to cardiometabolic diseases and dyslipidemia. Despite the preponderance of research about microbial interactions with these lipids, lacking are molecular insights into how the interactions occur and how they are regulated. We will address this knowledge gap, which we posit will reveal novel targets for therapeutic interventions in bacterial colonization and aberrant sterol lipid metabolism. Given that some bacteria produce sterols de novo, we reasoned that achieving an understanding of sterol handling in bacteria that make them could reveal insights into their handling in bacteria that use them. We therefore focused on Methylcoccus capsulatus, a bacterium reported to produce sterols nearly 40 years ago. Recent studies reported a significant divergence in sterol biosynthesis in M. capsulatus. We have since added to those reports one showing that sterol trafficking is also substantially different. We identified three proteins that traffic sterols: BstA, BstB, and BstC. BstA is a member of the resistance nodulation division family of transporters that work as transporters for a wide range of bacterial metabolites. BstB is a periplasmic binding protein with homologs involved in phosphonate transport. Finally, BstC is an outer membrane associated lipoprotein belonging to a family of transporters whose substrates are not known. The overall structures of the Bst proteins are markedly different from eukaryotic sterol transporters. However, they all contain ligand sites that are similar in the presentation of hydrophobic and hydrophilic residues. We posit that a modified structural genomics approach wherein the focus is on ligand sites instead of overall structure/sequence would enable the identification of functionally homologous proteins in bacteria. This work will use bioinformatics, quantitative ligand binding analyses, and structural approaches to identify and characterize sterol trafficking proteins in bacteria that make sterols, pathogens that hijack sterols, and gut flora that modulate host sterol metabolism.